Age-Related Pregnancy Results and Further Examination of Bitches after Aglepristone Treatment of Pyometra

Reprod Dom Anim doi: 1.1111/j.1439-531.28.1288.x ISSN 936-6768 Age-Related Pregnancy Results and Further Examination of Bitches after Aglepristone Treatment of Pyometra P Jurka 1, A Max 1, K Hawryn ska 1 and M Snochowski 2 1 Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw; 2 The Kielanowski Institute of Animal Physiology and Nutrition, PAN, Jablonna, Poland Contents The cystic endometrial hyperplasia and pyometra complex is one of the most common uterine diseases in bitches. The appearance of pharmacological preparations containing antiprogestagens created new possibilities for pyometra treatment. The aim of this study was to evaluate the curative effect of the anti-progestagen aglepristone treatment of pyometra in bitches of different ages. Twenty four bitches of different breeds, aged from.8 to 9.5 years (21 48 kg) exhibiting clinical pyometra symptoms (two groups I 5 years, n = 14 and II >5 years, n = 1) were evaluated. Information about the general reproductive health was collected up to 54 months after antiprogestagen treatment. Remission of clinical symptoms and return of blood chemistry results and total leucocyte count to referential values were achieved in all cases within 14 days of treatment. Bitches were naturally mated at the first, and when unsuccessful, the second oestrus after treatment. In group I, no recurrence of pyometra symptoms was observed during following cycle(s). Eight bitches (57.1%) had a full-term pregnancy and the number of newborn pups ranged from 1 to 12. None of the bitches from the group II became pregnant. In conclusion, the basic indication for conservative pharmacological treatment of pyometra is preserving female fertility and obtaining offspring. The important conditions for successful aglepristone treatment are: the young age (up to 5 years) and the lack of detectible ovarian cysts. It seems necessary to mate bitches in the first or second oestrus after finishing treatment. The efficacy of treatment can be measured by the after-treatment pregnancy rate. Introduction The cystic endometrial hyperplasia and pyometra complex (CEH P) is one of the most common uterine diseases in bitches. It is progesterone-dependent and occurs in the first half of the diestrous stage when blood progesterone concentration is raised and oestradiol concentration is at basal level. Ovariohysterectomy is the most common method of its treatment (Trasch et al. 23; Fieni 26). Surgery excludes the risk of recurrence of disease, but is connected with the loss of breeding potential. In several situations, owners refuse surgical treatment. Currently, pyometra is treated medically by applying broad-spectrum antibiotics, prostaglandin F 2a, ergot alkaloids, and anti-progestagens (Trasch et al. 23; Gu rbulak et al. 25). Many different antimicrobials have been used successfully, but in vitro sensitivity studies and clinical evidences suggest that amoxycillin, amoxycillin combined with clavulanic acid, cephalosporins or potentiated sulfonamides are good drugs of choices. It is recommended that the antimicrobial therapy should be continued minimum for 1 14 days (Verstegen and Verstegen-Onclin 26). Prostaglandin F 2a (either natural or synthetic) is proved to be useful in treatment of pyometra because of its luteolytic effect causing decrease in blood progesterone concentration as well as stimulation of the uterine contractility, but its main disadvantage are serious side effects (Gobello et al. 23; Trasch et al. 23; Fieni 26). Fieni et al. (21) successfully treated bitches with pyometra using combination of aglepristone and cloprostenol, but 68% of them showed different side effects. To avoid or minimize those side effects, prostaglandins are applied in combination with ergot alkaloids or anti-progestagens, which allow using the minimal dosage (Gobello et al. 23; Fieni 26). Anti-progestagens create new possibilities for treatment of pyometra, helping to restore the functions of the reproductive system in bitches (Blendinger et al. 1997; Gobello et al. 23; Trasch et al. 23). A few antiprogestagens causing an anti-progesterone effect may be taken into consideration as curative in pyometra cases: mifepristone (RU38486), onapristone (ZK98299) and aglepristone (RU46534). Mifepristone and aglepristone have been tested in animal reproduction, but only aglepristone is registered for veterinary use. Aglepristone is a synthetic steroid, which binds to progesterone receptors and prevents progesterone from inducing its biological effects. It binds to uterine progesterone receptors with affinity three times greater than progesterone itself in bitches and nine times greater in queens. Aglepristone not only blocks free receptors but also displaces progesterone already bound to receptors (Hoffmann and Schuler 2). Aglepristone has been used for induction of abortion and was also proved to cure other progesterone-dependent diseases. In addition to pregnancy termination, aglepristone is useful in conservative treatment of pyometra in bitches and queens, induction of parturition, treatment of mammary fibroadenomatosis in queens (Go rlinger et al. 22) and vaginal fibroma in bitches (Rollo n et al. 28). Aglepristone s mechanism of action consists of blocking the biological effect that progesterone exerts on cells or tissues. Aglepristone does not modify plasma concentrations of progesterone, but indirectly induces uterine contractions and cervix dilation, which stimulates emptying of the uterine cavity (Lennoz-Roland 25). Although aglepristone is considered a safe drug, it is not recommended for animals with renal or hepatic dysfunctions, in case of diabetes, general bad condition and suprarenal glands insufficiency. Several side effects have been observed after the administration of aglepristone such as anorexia, restlessness, depression,

2 P Jurka, A Max, K Hawryn ska and M Snochowski vomiting, diarrhoea, decrease in body temperature and local inflammatory reaction after injection (Breitkopf et al. 1997; Fieni 26). There are many different clinical protocols for therapy using anti-progestagens. Effectiveness of treatment may differ, as judged from published reports. The protocol advised by the experts meeting (Lennoz-Roland 25) as a method of pyometra treatment is as follows: subcutaneous injection of 1 mg kg s.c. of aglepristone then repeated at days 2, 7 and 14 and 28, if necessary. This may be accompanied by administration of antibiotics and or prostaglandins (Gobello et al. 23; Trasch et al. 23; Wehrend et al. 23; Gu rbulak et al. 25; Fieni 26). Severity of disease and treatment efficacy are evaluated not only on the basis of clinical signs but also through haematological and biochemical indicators. Total leucocyte count is widely accepted indicator of inflammation and its recovery, and is a subject to dynamic changes. Transaminases are commonly measured as a part of diagnostic liver function tests. Serum creatinine and blood urea nitrogen are most commonly used indicators of renal function. A rise in their concentration in blood indicates kidney damage or insufficiency. The aim of this study was to evaluate the curative effect of the anti-progestagen aglepristone treatment of pyometra in bitches of different ages. The dependence of age and the success rate of conservative treatment were observed. This was measured by successful full-term pregnancy in two following oestrous cycles after treatment and by remission of clinical symptoms of pyometra. Materials and Methods Twenty four bitches with CEH P of different breeds aged from.8 to 9.5 years (21 48 kg) were evaluated. During the study, animals were divided into two groups, according to their age. included 14 animals up to 5 years of age (3.46 ± 1.28), the remaining 1 animals older than 5 years (7.28 ± 1.13) were included in group II. All bitches were examined clinically including abdominal ultrasonography. The treatment began at week 2 4 of diestrus by single injection of 1 mg kg s.c. of aglepristone (Alizine, Virbac, Carros, Cedex, France) repeated at days 2, 7 and 14. During the first 7 days, daily injections of 8.75 mg kg of amoxicillin + clavulanic acid (Synulox, Pfizer, Sandwich, UK) were also given. Clinical examination was made every 7 days up to 21 days after the treatment started. Blood samples were collected from supraradial vein on day 1, 7 and 14. Total leucocytes count, concentrations of aspartate transaminase, alanine transaminase, alkaline phosphatase, creatinine, blood urea nitrogen, progesterone and oestradiol (on day 1 only) were measured. The number of leucocytes was determined using automatic analyser: Cell-dyn 17; Abbott, Los Angeles, USA 1997. The analytical kits were used for determination of blood level of aspartate transaminase, alanine transaminase, alkaline phosphatase, creatinine and blood urea nitrogen (Pointe Scientific, Warsaw, Poland). The concentration of progesterone in blood was analysed by chemiluminescence immunoassay (Immulite, Diagnostic Products Corp., Los Angeles, CA, USA). All analyses were performed twice. The efficiency of extraction ranged from 92% to 99%, minimum value and interassay variation were.5 ng ml and 8. % respectively. Determination of oestradiol concentrations was performed using immunofluorescent method and immunoenzymatic kits for quantitative determination oestradiol (Pointe Scientific, Warsaw, Poland), preceded by extraction with ethyl acetate. All analyses were duplicated. The efficiency of extraction ranged from 92% to 99%, minimum value and interassay variation were 1 pg ml and 9.6 % respectively. The diameters of uterine horns were measured using abdominal ultrasonography. The measurements of the uterine horn diameter as well as the monitoring of uterine changes were performed using Echoson Desmin VET (Echoson, Pulawy, Poland) and 5. 7.5 MHz linear transducer. The short-term success of the treatment was defined up to day 21 after the start of the treatment. All animals were primarily designed for natural mating at the first and when unsuccessful, the second oestrus after treatment. All animals underwent further examination which took place during next two oestrous cycles approximately 6 and 12 months after the treatment. Information about the general reproduction health in bitches up to 54 months after anti-progestagen treatment was collected by questioning owners over the phone. Statistical analysis all numerical data are expressed as mean ± SD with comparisons between groups performed by simple Student s t-test analysis using the program Statistica for Windows (Statistica 5., StatSoft, Cracow, Poland): p-value was set at.5. Results General information Of all 24 animals that were included in the study, 16 were in general good condition (67%) and the remaining eight bitches were in general bad condition (33%). In three cases, oestrogens were used in the past for prevention of nidation (12.5%). In one bitch, oestrogens were applied 8 weeks before the start of the treatment and in remaining two cases, they were used 6 weeks before the treatment began. In one case, progestagens were regularly used to post-pone the cycle (4.17%). Following clinical symptoms were observed: polyuria and polydypsia in 14 cases (58.3%), limited appetite or anorexia in 15 cases (62.5%), fever in six cases (25%), vomiting in two cases (8.3%), vaginal discharge was observed in four cases (16.7%). Initial vaginal discharge was observed 24 h after first administration of aglepristone in four animals. In the remaining cases, emptying of uterus began within 36 48 h after the treatment started. USG examination At the beginning of the treatment, there were no statistical differences in average diameters of uterine

Age-Related Pregnancy Results 3 horns measured in ultrasonographic examination between group I (3.93 ±.93 cm) and group II (3.37 ±.41 cm) (Fig. 1). After 14 days of treatment, the diameter of uterine horns was 1.26 ±.21 cm in group I and 1.47 ±.42 in the group II (p >.5). On day 14 of treatment, the uterine diameters of both groups were lesser than that on day 1 (p.1). In ultrasonographic examination performed at that time, cystic endometrial hyperplasia was not diagnosed. Blood analysis In both groups of examined animals leucocytosis was observed, in group I (29.5 ± 4.59 1 9 l) and in group II (31.4 ± 3.59 1 9 l). After 14 days of treatment in both groups of animals, the number of leucocytes was near reference values (Fig. 2). Serum concentration of aspartate transaminase and creatinine in both groups of animals was higher during the first 7 days of treatment and decreased to referential values after 14 days (p >.5) (Fig. 3 and 4). Concentrations of alanine transaminase, alkaline phosphatase, blood urea nitrogen were not higher than referential values through the whole period of treatment. The animals of group I and II were characterized by initial concentration of progesterone (P 4 ) of 16.4 ± 3.8 ng ml and 27.4 ± 4.8 ng ml respectively (p >.5). The mean P 4 level did not change significantly in time. Initial estradiol concentration in animals of group I and group II was 24.6 ± 4.9 pg ml and 37.3 ± 8.76 pg ml respectively (p >.5). Interestrous period The length of interestrous period before the treatment was determined on the basis of anamnesis. It was approximately 6 months in group I and 7 months in group II. Based on that, the expected time of oestrus after treatment was determined. In group I oestrus begun at the expected time in 8 14 cases (181 ± 8 days) and in the remaining six animals, interestrous period was shortened to 149 ± 11 days (p <.5). In group II, after treatment in 6 1 cases, oestrus started at expected time (198 ± 14 days), while in four remaining animals, interestrous period was shortened to 166 ± 15 days (p <.5). Pregnancy results No recurrence of pyometra symptoms was observed during the observation period in animals from group I. All the bitches in group I were mated and 8 14 (57.1%) had a full-term pregnancy and the number of newborn pups ranged from 1 to 12 (one puppy born by 5 year old bitch). In group II, nine out of ten animals were mated in the first oestrus after treatment and none of them became pregnant. Three bitches from this group experienced relapse in the next oestrus after treatment and were treated surgically with ovariohisterectomy. In those cases, ovarian cysts and cystic endometrial hyperplasia were diagnosed during the operation. Another animal underwent the ovariohisterectomy because of owner s 5 4 3 2 1 Diameter of uterine horn (cm) I 6 4 Aspartate transsminase (IU/l) I 2 1 7 14 Fig. 1. Diameter of uterine horn during aglepristone treatment of pyometra in bitches of various ages Fig. 3. Concentration of aspartate transaminase during aglepristone treatment of pyometra in bitches of various ages 4 Leucocytes ( 1E9/l) I 2 Creatinine (mg/dl) I 2 1 Fig. 2. Total leucocytes count dynamics during aglepristone treatment of pyometra in bitches of various ages Fig. 4. Concentration of creatinine during aglepristone treatment of pyometra in bitches of various ages

4 P Jurka, A Max, K Hawryn ska and M Snochowski decision. Five bitches were mated again in the second oestrus. Three of them were treated surgically 2 weeks after the second oestrus according to owners wishes, although no recurrence of pyometra appeared in those cases. In two remaining cases, no data were collected after the second oestrus. Long-term observation During long-term observation, data were collected about 13 animals from group I, in eight cases up to 36 months after treatment, and in five bitches up to 54 months after treatment. Additional pregnancies finished with parturition in three bitches (in one animal twice), which previously delivered puppies after aglepristone treatment. Animals from group II were not subject to long-term observation, for most of them were treated with ovariohisterectomy before. Discussion During the last 1 years, only a few investigations on the use of aglepristone in pyometra treatment were undertaken and published. In different studies, most of the animals examined were not mated in the next oestrus, and there is few information about the possibility of receiving successful pregnancy after aglepristone treatment (Gobello et al. 23; Trasch et al. 23; Gu rbulak et al. 25; Fieni 26). No research was made showing the dependence of the age of treated bitches and the rate of successful pregnancy, and none of the available studies describes the effect of pregnancy on the recurrence rate of pyometra in long-time perspective ( 1 year). In this study, in bitches from group I, the rate of successful pregnancy was 57.1% and none bitches from group II became pregnant. Authors suggest that this may be because of more serious damage to endometrium in older animals. Furthermore, age-related changes in fertility include increased interestrous interval, decreased conception rate and decreased litter size were observed in bitches (Johnston et al. 21). No research has been performed investigating the dependence between the age of animals and the course of emptying of the uterus during aglepristone treatment. In this study, the emptying of uterus was evaluated using USG examination. In both groups, this process took similar course, which is proved by the lack of statistically important differences between the diameters of uterine horns in both groups after 14 days (Fig. 1). In this study, in both age groups, the opening of uterine cervix began within 24 48 h after administration of aglepristone, which was comparable to the results obtained by other authors (Breitkopf et al. 1997; Wehrend et al. 23; Gu rbulak et al. 25; Fieni 26). In the literature, the relation between the age of treated bitches and the appearance of relapse during long period of time has not been investigated. Frequency of recurrence in different researches alternated between 18.9% and 2% (Gobello et al. 23; Trasch et al. 23; Fieni 26). In all the studies, only few animals were observed to the beginning of the next oestrus and very few were mated in the following cycle. In this study, no recurrence of pyometra was observed in young animals (group I), while in three cases (3%) of older bitches (group II), pyometra reoccurred after the first oestrus after treatment. In those three cases, the initial estradiol level was 45.53 ±.58 pg ml, which was significantly more than the mean concentration for the remaining animals in this group, 34.8 ± 4.21 pg ml (p <.1). In these bitches, ovarian cysts were diagnosed during operation. Ultrasonographic diagnosis of ovarian cysts in some cases is possible. Yet, it is not possible to differentiate by ultrasonography between oestrogen-producing ovarian cysts, clinically irrelevant paraovarian cysts and corpora lutea with a fluid filled cavity (Blendinger and Bostedt 1991; Tammer et al. 1994; Lemmer 1999; Wehrend and Bostedt 22). Thus, the high concentration of oestradiol is the factor which should be taken into consideration when evaluating the risk of recurrence. Recurrence rate in group II was higher than that reported by authors quoted above, which may indicate higher proneness to recurrence in older bitches treated with aglepristone, but has to be confirmed on numerous clinical materials. In biochemical indicators, no statistical differences were observed between measurements made on particular days of treatment or between the two groups. Yet, the concentration of aspartate transaminase at the beginning of the treatment was above the referential values in both groups (Fig. 3), which may indicate the liver dysfunction caused by circulation of bacterial toxins originating from uterus. This corresponds to the results presented by England et al. (27) and De Schepper et al. (1987). Blond concentration of creatinine in animals from group II at the beginning of the treatment was slightly above the referential values (Fig. 4). This result may be because of the age of the examined animals and the lower renal sufficiency during pyometra. In our research, the healing rate (ceasing of clinical signs) of conservative treatment with aglepristone is noticeably higher than that reported by other authors (Fieni et al. 21; Trasch et al. 23). This may be caused partially by limited number of animals involved in the study and by the selection of animals (ovariohisterectomy was performed in cases when owners refused conservative treatment). Interestrous period was shortened after aglepristone treatment in six (43%) and four (4%) animals from groups I and II respectively, which is slightly more than that reported by Trasch et al. (23). Results of long-term observation indicate that at least in some of young animals, fertility may be preserved for a long time after aglepristone treatment. In older animals, treatment may be used as a preparation for surgical treatment ovariohisterectomy. We conclude that the basic indication for conservative pharmacological treatment of pyometra is preserving female fertility and obtaining offspring. The important conditions for successful aglepristone treatment can be summarized as the young age (up to 5 years) and the lack of detectible ovarian cysts. It seems necessary to mate bitches in the first or second oestrus after finishing treatment. The efficacy of treatment can be measured by the after-treatment pregnancy rate.

Age-Related Pregnancy Results 5 Author contributions Jurka designed the study, analyzed data and wrote paper, Max analyzed data and wrote a part of paper, Hawryńska was involved in study design and analysed data and Snochowski analyzed data. References Blendinger K, Bostedt H, 1991: Zum alter und Zyklusstadium bei Hu ndinnen mit pyometra. Tiera rztl Prax 19, 37 31. Blendinger K, Bostedt H, Hoffmann B, 1997: Hormonal state and effects of the use of an antiprogestin in bitches with pyometra. J Reprod Fertil Suppl 51, 317 325. Breitkopf M, Hoffmann B, Bostedt H, 1997: Treatment of pyometra (cystic endometrial hyperplasia) in bitches with an antiprogestin. J Reprod Fertil Suppl 51, 327 331. De Schepper J, van der Stock J, Capiau E, 1987: The characteristic pattern of aspartate aminotransferase and alanine aminotransferase in the bitch with the cystic hyperplasia-pyometra complex: effect of medical or surgical treatment. Vet Res Commun 11, 65 75. England GC, Freeman SL, Russo M, 27: Treatment of spontaneous pyometra in 22 bitches with a combination of cabergoline and cloprostenol. Vet Rec 16, 293 296. Fieni F, 26: Clinical evaluation of the use of aglepristone, with or without cloprostenol, to treatment cystic endometrial hyperplasia-pyometra complex in bitches. Theriogenology 66, 155 1556. Fieni F, Bruyas JF, Tainturnier D, Battut I, 21: Clinical use of antigestagens in the treatment of metritis pyometra in the bitch. EVSSAR Newslett 1, 13 14. Gobello C, Castex G, Klima L, Rodriguez R, Corrada Y, 23: A study of two protocols combining aglepristone and cloprostenol to treat open cervix pyometra in the bitch. Theriogenology 6, 91 98. Go rlinger S, Kooistra H, van den Broek A, Okkens A, 22: Treatment of fibroadenomatous hyperplasia in cats with aglépristone. J Vet Intern Med 16, 71 713. Gu rbulak K, Pancarc SM, Ekc H, Konuk C, Krsan I, Ucmak M, Toydemr S, 25: Use of aglepristone and aglepristone + intrauterine antibiotic for the treatment of pyometra in bitches. Acta Vet Hung 53, 249 255. Hoffmann B, Schuler G, 2: Receptor blockers general aspects with respect to their use in domestic animal reproduction. Anim Reprod Sci 6 61, 295 312. Johnston SD, Root Kustritz MV, Olson PNS, 21: Canine and Feline Theriogenology. W. B. Saunders Company, Philadelphia, pp. 26 224, 269. Lemmer W 1999: Untersuchen zur konservativen Pyometrabehandlung der Hu ndin mittels eines Antigestagens im Rahmen einer offenen klinischen Studie. Diss Med Vet Gießen, pp. 124 125. Lennoz-Roland M, 25: Villefontaine France. Practical use of aglepristone review of experts meeting. Nice, 17-18.6.25, 7 8. Rollo n E, Millán Y, de las Mulas MJ, 28: Effects of aglepristone, a progesterone receptor antagonist, in a dog with a vaginal fibroma. J Small Anim Pract 49, 41 43. Tammer I, Blendinger K, Sobiraj A, Bostedt H, 1994: U ber den Einsatz der exfoliativen Vaginalzytologie im Rahmen der gynäkologischen Befunderhebung bei der Hu ndin. Tiera rztl Prax 22, 199 27. Trasch K, Wehrend A, Bostedt H, 23: Follow-up examinations of bitches after conservative treatment of pyometra with the antigestagen aglepristone. J Vet Med A, 5, 375 379. Verstegen J, Verstegen-Onclin K 26: Pyometra in the bitch and queen. In: Proceedings, NAVC Conference (Eds), NAVC, IVIS, Ithaca, NY. Available at: http://www.tnavc. org; http://www.ivis.org. Wehrend A, Bostedt H, 22: Zur Bedeutung und Behandlung des Ovarialzystensyndroms bei der Hu ndin: Proceedings. 48.Jahrestagung der FK-DVG vom 3.8. bis 1.9.22, Blackwell Wissenschafts-Verlag GmbH, Magdeburg, 261 264. Wehrend A, Trasch K, Bostedt H, 23: Treatment of the closed type of pyometra by the antigestagen, aglepristone, in the bitch. Kleintierpraxis 48, 679 683. Submitted: 19 Sep 28 Author s address (for correspondence): Dr Piotr Jurka, Faculty of Veterinary Medicine, Department of Clinical Sciences, Warsaw University of Life Sciences, Nowoursynowska Str. 166, 2 787 Warsaw, Poland. E-mail: piotr_jurka@sggw.pl