Vet. Pathol. 18:208-218 (1981)

Involution Abnormalities in the Postpartum Uterus of the Bitch M.A. AL-BASSAM, R. G. THOMSON, and L. O'DONNELL Department of Pathology and Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Abstract. Of 95 reproductive tracts from postpartum bitches, 20 had subinvolution of the placental sites. Grossly, the placental sites were hemorrhagic and about twice the size of normal sites at the same time after parturition. Microscopically, large masses of collagen, hemorrhage and dilated endometrial glands were in the endometrial sites. The myometrium was invaded in many areas by trophoblast-like cells from the endometrium. Variations in the size of placental sites in the same uterus were found in eight cases, and histological variations in the involution process in five. Invasion of the myometrium by trophoblast-like cells four days after parturition was seen in one bitch.

Subinvolution of placental sites in a bitch was reported in 1966 151. This condition was distinguished from others that cause hemorrhage from the genital tract, such as cystitis, tumors of the bladder, trauma, metritis, endometrial hyperplasia and tumors of the genital tract. Other reports have described the condition in more detail [9, 31. The pathogenesis and cause of subinvolution are unknown. Hormonal influences [9, 141 and the invasion of the endometrium and the myometrium by trophoblast-like cells 191, however, have been considered to be factors in preventing normal involution. There is one report of spontaneous recovery (201. Materials and Methods The 95 reproductive tracts examined were from a study of normal postpartum involution of the canine uterus [l]. Reproductive tracts were obtained from postpartum spayed bitches and experimental surgical cases. Seventy-five reproductive tracts were obtained from dogs of different breeds and ages, with or without history of uterine disease. Fifty tracts were from dogs up to three months after parturition, 10 from dogs more than three months after parturition, 12 were at different stages of pregnancy, and three had pyometra. All tracts were fmed in 10% formalin. Twenty pregnant or recently whelped bitches of different ages and breeds were obtained for experimental procedures. Uterine samples were taken at specific times. From the first four dogs, four to six uterine samples were taken surgically at different intervals after parturition. Only two surgical procedures were done in the others: in the first procedure, one uterine horn was taken, and in the second, the other horn and two ovaries were taken. Forty-six uterine samples were obtained surgically from dogs between one and eight weeks after whelping. In each specimen, the following were recorded: the number and size of placental sites, gross 208

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Fig. 1: Uterine horn, serosal surface from bitch six weeks after parturition. Large ellipsoidal swelling; diameter of largest areas about twice normal size. Fig. 2 Same uterine horn as in fig. I . Mucosal surface contains small amounts of bloodtinged mucus; placental sites irregularly thick, rough and dark; small areas of hemorrhage.

changes in each placental site, and the number and size of corpora lutea. From the first four dogs, only one placental site was removed at each time interval. From the others, at least three placental sites in each uterine sample were selected, and transverse or longitudinal pieces were taken from the placental sites and interplacental uterine tissue. The trimmed tissues were embedded in paraffin, sectioned at 5 ,urn and stained with hematoxylin and eosin (HE). Masson’s trichrome stain [ 161 was used to determine the presence of collagen and fibrin. Before fixation, uterine swabs for bacterial cultures were taken from 10 of the surgical specimens. Ten milliliters of blood were collected at surgery from the cephalic vein into evacuated tubes containing 143 USP units of sodium heparin solution. Plasma progesterone was determined in 10 samples by a competitive protein binding assay [17, 181 after a rapid method of sample preparation [12]; radioimmunoassay [6] was used in the remaining 12 samples.

Results Of the 95 tracts examined, 27 had significant abnormalities of placental sites. Twenty bitches had subinvolution of the placental sites at different intervals after parturition. The diagnosis was based on the history and on gross and histologic examination of the placental sites. The usual history was persistent bloody vaginal discharge. On gross examination, affected placental sites appeared from the serosal surface as large ellipsoidal swellings; the diameter of the largest was about twice the size of a normal site from the same breed at the same stage (fig. 1). The lumen of the

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Fig. 3: a. Diagram of subinvolution with invasion of myometrium, retention of collagen and masses and dilation of glands. b. Similar subinvolution but with surface necrosis and much hemorrhage.

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Fig. 4: Syncytial trophoblast-like cells in subinvolution of placental sites seven weeks after parturition. Myometrium invaded by syncytial trophoblast-like masses. Few muscle fibers between cells. Some trophoblast-likecells vacuolated.

uterus contained a small amount of blood-tinged fluid and the placental sites were irregularly thick, rough, and gray to brown, with areas of hemorrhage of various sizes (fig. 2). The interplacental endometrium was smooth, glistening and gray-tan. Histologic diagnosis depended on large amounts of collagen without massive sloughing by 12 weeks after birth. The surface of the lobulated collagen masses was necrotic and hemorrhagic. This collagen mass extended down to involve the whole mucosa or even part of the myometrlum (fig. 3). Another criterion for diagnosis was that the endometrial glands in the mucosa were greatly dilated, fewer than normal and surrounded by more collagen than normal. The mucosa looked like a thick eosinophilic mass interlaced with a few dilated glands. Diagnosis also depended on individual trophoblast-like cells (or decidual cells) in abundant numbers in the base of the collagen masses in the area of invasion. A few syncytial masses of the same cells had invaded into the muscle layers (fig. 4). Many of these cells extended to the myometrium and surrounded the blood vessels in the stratum vasculare (fig. 3, 5). In some areas the collagen tissue was interlaced with large sinusoidal spaces and scattered hemorrhages of various sizes (fig. 3). Mononuclear cells were absent, especially in areas where the trophoblast-like cells had invaded the myometrium (fig.

3). Slightly more than two-thirds of the cases were in bitches under 3 years old and from 7 to 12 weeks after whelping. There was no breed predisposition. The progesterone from three dogs was low, 0.5, 1.3, and 2.4 ng/ml respectively,

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Fig. 5: Blood vessel in stratum vasculare of myometrium at placental sites from bitch with subinvolution of placental sites, seven weeks after parturition. Trophoblast-like cells oriented around blood vessel; only endothelium remains intact from vessel wall.

and no difference was found between these and normal dogs at the same time after parturition. The level in the last case was from a bitch six weeks after parturition; the same bitch had a high progesterone level, 9.2 ng/ml, four days after parturition. Variations in size and thickness of the placental sites were noted in eight cases (fig. 6). Some of the placental sites were up to two times larger than normal, and were rough and gray-brown with hemorrhagic areas of various sizes; other sites in the same uterus were smaller, finely granular or even completely involuted. The involuted sites were smooth, dark brown, narrow bands. Subinvolution of some of the placental sites was diagnosed histologically in five uteri, whereas other placental sites in the same uterus had normal involution (fig. 7, 8). Invasion of the myometrium by trophoblast-like cells was seen only in one uterus four days after parturition. These cells started from the endometrium and grew down into the myometrium in a wedge. Many of these cells were around the small blood Fig. 6 Schematic drawing of eight uteri at different times after parturition; gross variations in shape and size between placental site attachment. a. Uterus 45 days, from 1 1-year-old King Charles Spaniel with no history of vaginal discharge or other reproductive problems. Histologically, large placental site had subinvolution, smaller placental sites in different stages of normal involution. b. Uterus 45 days, from 7-year-old German Shepherd with no history of vaginal discharge. Histologically, selected sections from placental sites had normal involution. c. Uterus 42 days, from 1-year-old Spaniel with history of hemorrhagic vaginal discharge since parturition. Histologically, two large placental sites subinvoluted, other sites normal. d. Uterus 60 days, from 14-month-old Irish Setter with no history of reproductive tract disease. Placental

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sites normal histologically. e. Uterus 28 days (age and breed not available) with no history of reproductive tract disease. Placental sites normal histological1y.f. Uterus 45 days, from 5-yearold Doberman Pinscher with history of persistent bloody vaginal discharge since parturition. Histologically, placental sites at top right and lower left normal, other sites subinvoluted. g. Uterus 56 days, from 13-month-old Labrador Retriever with no history of reproductive tract disease. Histologically, top placental site on each side subinvoluted, lower ones on each side normal. h. Uterus 70 days, from 6-year-old Siberian Husky with no history of reproductive tract disease. Histologically, top right site subinvoluted; involution in bottom right placental site almost complete; site at lower left normal. 213

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Fig. 7: Endometrium: much collagen, glands, and mononuclear cells; myometrium invaded by a few trophoblast-like cells.

vessels in the area of invasion. They were multinucleated, with basophilic, highly vacuolated cytoplasm. These cells could be retained fragments of normal trophoblastic cells of pregnancy (fig. 9). Three uteri had acute postpartum endometritis. Two were in the first week and one in the second week. The diagnosis depended on the bacteriological and histological examination in one case, and on the histological examination in the other two cases. Bacterial culture of the uterus yielded Staphylococcus uureus in large numbers. No bacteria were cultured from nine other uteri. No significant differences were seen between the interplacental endometrium and ovaries from the subinvolution uteri and from normal uteri at the same time postpartum. Discussion The invasion of the endometrium and myometrium four days after birth by trophoblast-like cells is important in the pathogenesis of subinvolution of the placental sites. In the pregnant woman, there is invasion of the myometrium by syncytial masses (so-called giant cells) of fetal trophoblasts. Such syncytial masses are found most frequently in the middle of pregnancy, but may persist until the end of gestation [4, 7, 10, 111. In the golden hamster, giant trophoblastic cells appear in the myometrium during gestation and up to three weeks after parturition [19]. The route of invasion is along the spiral uterine arteries both in man and golden hamster. Such retained fragments of normal trophoblastic cells die out promptly and spontaneously after abortion and full-term or abdominal pregnancy [4]. Our study of the canine uterus shows the same route of invasion, and then degeneration of the retained fragments of normal trophoblast-like cells seems to occur in the normal postpartum endometrium and myometrium.

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t

Fig. 8 Endometrium almost involuted with sloughed collagen mass in lumen, many mononuclear cells in lamina propria.

For unknown reasons, the retained trophoblastic cells did not regress or degenerate, but continued to invade the deep glandular layer or even the myometrium. The retention of these cells for a long time after birth is an important factor in the prevention of the normal involution process. These findings are in agreement with previous observations [9]. One previous report indicated that decidual cells invaded to the myometrium [ 131. We cannot rule out the possibility of decidual cells in the cases of subinvolution, because it is difficult to identify them by light microscopy [2]. Three possible functions have been ascribed to the decidua in the human pregnant uterus: nutritional, protective and parturitional [ 151. Another possible function, control of trophoblastic infiltration, may explain the persistence of differentiated decidual cells in subinvolution. Our study offers evidence that the invasion is trophoblastic, and the decidual cell differentiation may then be a secondary response to the invasion of trophoblast-like cells. Noninvolution of the placental site in man is accompanied by failure of occlusion of the vessels in the exposed placental bed and is manifested clinically by hemorrhage [8]. The defective vessels contain poorly attached thrombi, often with little organization. Dissolution of mural constituents of these vessels associated with trophoblastic invasion of the endometrium during early gestation may impair contraction after delivery [8]. The failure of thrombosis and occlusion of the endometrial blood vessels

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Fig. 9: Myometrium four days postpartum, invaded by trophoblast-like cells. Many cells around blood vessels multinucleated with basophilic to highly basophilic cytoplasm.

in the canine subinvoluted placental site is considered an important cause of hemorrhage, and may be caused by damage to vessels by the trophoblast-like cells. Differencesin degree of involution between placental sites in the same uterus indicate that hormonal influence and bacterial infection probably are not major factors in the pathogenesis of the subinvolution. It is possible that subinvolution usually is not seen after the 12th week because most of the cases are noticed between the 7th and 12th weeks and ovariohysterectomyusually is done on such dogs. Also, the condition may resolve without surgical intervention. One case report [20] indicated that recovery from subinvolution of the placental sites is possible without surgical intervention, but the histologic picture showed sloughing of collagenous masses into the lumen and repaired endometrium in the area. These changes indicate that the involution process was active and suggest to us that the condition was delayed normal involution. Treatment of the condition other than by ovariohysterectomy generally has not been successful [3, 9, 141. One clinical report [3] indicated that a fast and successful treatment of postpartum uterine bleeding and subinvolution of placental sites was injection of a single dose of 25 to 50 mg of medroxyprogesterone. That author considered that the bleeding was due to an increase in capillary permeability in the endometrium or decreased clotting time induced by estrogen, which can be prevented

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by progesterone. The author depended solely on the absence of hemorrhage to indicate a cure of the condition. Others have said that progesterone may be a cause of subinvolution [9, 141. Our work did not demonstrate the significance of progesterone as a factor in preventing postpartum involution of the uterus in the dog. Factors other than hormones may predispose to subinvolution. The precise pathogenesis of the condition still is not clear. Acknowledgements We thank Michael Baker-Pearce for technical assistance, Ted Eaton, Ryan Van Vliet for photography and Ms. Valerie Manos for technical drawings.

References 1 AL-BASSAM, M.; THOMSON, R.G.; ODONNELL, L.: Normal post partum involution of the uterus in the dog. Can J Comp Med (submitted) 2 ANDERSON, J.W.: Ultrastructure of placenta and fetal membranes of the dog. Anat Rec 165 15-36, 1969 3 ARBEITER, K.: The use of progestins in the treatment of persistent uterine hemorrhage in the postpartum bitch and cow. A clinical report. Theriogenology 4 11-13, 1975 4 BAGSHAWE, K.D.: Normal trophoblast in choriocarcinoma. In: The Clinical Biology of the Trophoblast and Its Tumors, pp. 9-27. Williams and Wilkins. Baltimore, 1969 5 BECK,A.M.; MCENTEE, K.: Subinvolution of placental sites in a post partum bitch. A case report. Cornell Vet 56:269-277, 1966 6 Bosu, W.T.K.; EDQVIST, L.E.; LINDBERG, P.; MARTINSSON, K.; JOHANNSON, E.D.B.: The effect of various dosages of lynestrenol on the plasma levels of estrogens and progesterone during the menstrual cycle in the rhesus monkey. Contraception 13677-684, 1976 7 BOYD,J.D.; HAMILTON, W.J.: The giant cells of the pregnant human uterus. J Obstet Gynaecol6R208-2 18, 1960 8 DRISCOLL, S.C.: Placental-uterine interrelationships. In: The Uterus, ed. Norris, Hertig, and Abell, pp. 213-226. Williams and Wilkins, Baltimore, 1973 9 GLENN,B.L.: Subinvolution of placental sites in the bitch. 18th Gaines Veterinary Symposium, pp. 7-10. Gaines Dog Research Centre, White Plains, N.Y., 1968 10 HAMILTON, W.J.; BOYD,J.D.: Trophoblast in human utero-placental arteries. Nature 212 906-908, 1966 1 1 HAMILTON, W.J.; MOSSMAN, H.W.: The implantation of the blastocyst and the development of the fetal membranes, placenta and decidua. In: Human Embryology, pp. 83-131. Williams and Wilkins, Baltimore, 1973 12 JOHANSSON, E.D.B.: Progesterone levels in peripheral plasma during the luteal phase of the normal human menstrual cycle measured by a rapid competitive protein binding technique. Acta Endocrinol61: 592-606, 1969 13 JUBB,K.V.F.; KENNEDY, P.C.: The female genital system. In: The Pathology of Domestic Animals, pp. 487-585, vol. I. Academic Press, New York. San Francisco, London, 1970 14 KIRK,R.W.; MCENTEE,K.; BENTINCK-SMITH, J.: Diseases of the urogenital system. In: Canine Medicine, ed. Catcott, pp. 3 8 7 4 8. American Veterinary Publications, Santa Barbara, California, 1968 15 MCLAREN, A.: Maternal factors in nidation. In: The Early Conceptus, Normal and Abnormal, ed. Park, pp. 22-23. D.C. Thomson and Co., Scotland, 1965 16 MCMANUS, J.F.A.; MOWRY, R.W.: The trichrome technique of Pierre Masson. In: Staining

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Methods, Histologic and Histochemical, pp. 234-236. Harper Medical Division, Harper and Row, New York, Evanston and London, 1965 MURPHY, B.E.P.: Application of the property of protein-binding to the assay of minute quantities of hormones and other substances. Nature 201:679-682, 1964 MURPHY, B.E.P.: Some studies of the protein-binding steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding radioassay. J Clin Endocrinol Metab 27:973-990, 1967 ORSINI,N.W.: The trophoblastic giant cells and endovascular cells associated with pregnancy in the hamster, Cricetus auratus. Am J Anat M273-331, 1954 SCHALL,W.D.; DUNCAN, J.R.; FINCO,D.R.; KNECHT, C.D.: Spontaneous recovery after subinvolution of placental sites in a bitch. J Am Vet Med Assoc 159 1780-1782, 1971

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