Effects of Aglépristone, a Progesterone Receptor Antagonist, Administered

Theriogenology 62 (2004) 494–500

Effects of agle´pristone, a progesterone receptor antagonist, administered during the early luteal phase in non-pregnant bitches S. Galaca,b,*, H.S. Kooistrab, S.J. Dielemanc, V. Cestnika, A.C. Okkensb aVeterinary Faculty, University of Ljubljana, Ljubljana, Slovenia bDepartment of Clinical Sciences of Companion Animals, Utrecht University, Yalelaan 8, P.O. Box 80154, TD Utrecht 3508, The Netherlands cDepartment of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Received 7 July 2003; received in revised form 28 October 2003; accepted 1 November 2003

Abstract

Agle´pristone, a progesterone receptor antagonist, was administered to six non-pregnant bitches in the early luteal phase in order to determine its effects on the duration of the luteal phase, the interestrous interval, and plasma concentrations of progesterone and prolactin. Agle´pristone was administered subcutaneously once daily on two consecutive days in a dose of 10 mg/kg body weight, beginning 12 1 days after ovulation. Blood samples were collected before, during, and after administration of agle´pristone for determination of plasma progesterone and prolactin concentrations. The differences in mean plasma concentration of progesterone and of prolactin before, during, and after treatment were not significant. Also, the duration of the luteal phase in the six treated bitches (72 6 days) did not differ significantly from that in untreated control dogs (74 4 days). However, the intervals during which plasma progesterone concentration exceeded 64 and 32 nmol/l were significantly shorter in the six treated bitches than in untreated control dogs. The interestrous interval was significantly shorter in beagle bitches treated with agle´pristone (158 16 days) than in the same group prior to treatment (200 5 days). It is concluded that administration of agle´pristone during the early luteal phase in the non-pregnant bitch affects progesterone secretion, but not sufficiently to shorten the luteal phase. The shortening of the interestrous interval suggests that agle´pristone administered in the early luteal phase influences the hypothalamic–pituitary–ovarian axis. # 2003 Elsevier Inc. All rights reserved.

Keywords: Progesterone receptor antagonist; Luteal phase; Prolactin; Interestrous interval; Dogs

* Corresponding author. Tel.: þ31-30-2539411; fax: þ31-30-2518126. E-mail address: [email protected] (S. Galac).

1. Introduction

Agle´pristone, a progesterone receptor antagonist, has proved to be safe and effective in early and midgestation pregnancy termination in the bitch [10,11,13]. Progesterone receptor antagonists terminate pregnancy by binding with high affinity to the uterine progesterone receptor, thereby preventing progesterone from exerting its biological effect [5,14]. In addition to pregnancy termination, endocrine effects such as temporary elevation of plasma prolactin concentration, premature cessation of the luteal phase, and shortening of the interestrous interval have been observed after administration of progesterone receptor antagonists [10,11,13,16]. However, depending on the time of administration, differences in the regulation of the luteal phase might cause other endocrine effects. So far none of the studies concerning the effects of progesterone receptor antagonists have been carried out early in the luteal phase in the cyclic non-pregnant dog. The regulation of canine luteal function in both cyclic and pregnant bitches differs between the first and the second parts of the luteal phase. Between ovulation and Days 24– 28 of the luteal phase, the corpora lutea function independently of pituitary support [18]. During the second part of the luteal phase pituitary luteotropic factors, such as prolactin and possibly LH, are necessary to maintain the luteal function [18–20]. The role of LH as a luteotropic factor remains unclear. It was reported in 1987 that LH may be necessary to sustain progesterone synthesis [3], but more recent studies failed to confirm a direct luteotropic function for LH in the bitch [19,21]. It was assumed that a progesterone receptor antagonist would have different endocrine effects early in the luteal phase in the non-pregnant cyclic bitch than it would in early pregnancy or midgestation and for this reason the effects of agle´pristone on plasma concentrations of progesterone and prolactin were studied in the early luteal phase, together with its effects on the length of the luteal phase and the interestrous interval.

2. Materials and methods

2.1. Animals, treatment, and collection of blood samples

Six healthy beagle bitches, 3–6 years of age, were housed singly or in pairs in indoor– outdoor runs, fed a standard commercial dry dog food once daily, and given water ad libitum. All dogs were examined three times per week for the presence of vulvar swelling and serosanguinous vaginal discharge, which were considered to signify the onset of proestrus. The first day of the luteal phase (Day 1) was defined as the day subsequent to the onset of proestrus on which plasma progesterone concentration exceeded 16 nmol/l, which is when ovulation is assumed to occur [2,28,17]. The luteal phase was considered to be terminated when plasma progesterone concentration was found to be below 3 nmol/l for the first time. The interestrous interval was defined as the period between the onset of one proestrus and that of the next. The bitches were treated with agle´pristone (Alizine1, Virbac Laboratories, Carros, France) in a dose of 10 mg/kg body weight subcutaneously, once daily on two consecutive days beginning 12 1 days after ovulation. Blood samples for measurement of plasma 496 S. Galac et al. / Theriogenology 62 (2004) 494–500 concentrations of progesterone and prolactin were collected at least three times weekly from the onset of proestrus to the end of the luteal phase. Blood samples were also collected immediately before and at 1, 2, 4, and 8 h after administration of agle´pristone. Blood samples were collected by jugular venipuncture, immediately placed in chilled heparin- coated tubes, and centrifuged. Plasma was stored at À208C until assayed. In three untreated control beagle bitches blood samples were collected once daily between the onset of proestrus and the 90th day of the luteal phase [19].

2.2. Radioimmunoassays

Plasma concentrations of progesterone were measured by a previously validated radioimmunoassay [6,17]. The intra-assay and inter-assay coefﬁcients of variation were 11 and 14%, respectively, and the lowest detectable level of progesterone was 0.3 nmol/l. Plasma concentrations of prolactin were measured by a previously validated heterologous radioimmunoassay [17]. The intra-assay and inter-assay coefﬁcients of variation were 3.5 and 11.5%, respectively. The lowest detectable level of prolactin was 0.8 mg/l.

2.3. Calculations and statistical analysis

The mean plasma concentrations of progesterone and prolactin 5 days before, during, and after treatment, until Day 21 of the luteal phase, were compared by ANOVA for repeated measures. The mean intervals during which plasma progesterone concentration exceeded 64 and 32 nmol/l in the group of bitches treated with agle´pristone were compared with those intervals in the control dogs using a one-tailed Student’s t-test. A two-tailed Student’s t-test was used to compare the mean duration of the luteal phase in the treated and control bitches and the mean interestrous interval in bitches before and after treatment with agle´pristone. Values are expressed as mean S:E:M: and/or range. P 0:05 was considered signiﬁcant.

3. Results

The administration of agle´pristone caused no side effects except a painless inflammation at the injection site in three bitches, which disappeared within 1 month. In the other three bitches the area was massaged after the injection and no inflammation was observed. The mean duration of the luteal phase in the six bitches treated with agle´pristone (72 6 days) did not differ from that in the untreated control bitches (74 4 days). Mean plasma progesterone concentration remained unchanged during the 2 days of treatment with agle´pristone. The mean plasma progesterone concentrations before treatment (106 13 nmol/l, range 70–140 nmol/l), during treatment (124 13 nmol/l, range 76– 156 nmol/l), and after treatment (130 16 nmol/l, range 89–184 nmol/l) did not differ. The mean interval during which plasma progesterone concentration exceeded 64 nmol/l in the treated bitches was 28 2 days, which was significantly shorter (P < 0:05) than that in the control bitches (34 3 days). The mean interval during which plasma progesterone concentration exceeded 32 nmol/l in the treated bitches was 39 2 days, which was S. Galac et al. / Theriogenology 62 (2004) 494–500 497

160 50

140 45 40 120 35 100 30 g/L) µ 80 25

60 20

15 prolactin (

progesterone (nmol/L) 40 10 20 5 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 (a) day of the luteal phase progesterone prolactin

160 50

45 140 40 120 35 100 30 g/L) µ 80 25

20 60

15 prolactin (

progesterone (nmol/L) 40 10 20 5

0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 (b) day of the luteal phase

progesterone prolactin

Fig. 1. Mean plasma concentrations of progesterone and prolactin in six beagle bitches treated with agle´pristone (a) and three control beagle bitches (b), starting from the day of ovulation (Day 1) to the end of the luteal phase. Agle´pristone was administered on two consecutive days, beginning 12 days after ovulation (arrows), subcutaneously in a dose of 10 mg/kg body weight, once daily. significantly shorter (P ¼ 0:05) than that (47 5 days) in the control bitches (Fig. 1). The interestrous interval in bitches treated with agle´pristone (158 16 days) was significantly shorter (P < 0:05) than in the same bitches before treatment (200 5 days). The profile of plasma prolactin concentrations in bitches treated with agle´pristone was characterized by a fluctuating baseline with occasional distinct elevations (Fig. 1). The differences in mean plasma prolactin concentration before treatment (3:2 1:1 mg/l, range 498 S. Galac et al. / Theriogenology 62 (2004) 494–500

1.3–8.7 mg/l), during treatment (3:2 0:8 mg/l, range 1.6–7.1 mg/l), and after treatment (3:2 0:8 mg/l, range 1.4–5.9 mg/l) were not signiﬁcant.

4. Discussion

Administration of agle´pristone in the early luteal phase did not influence plasma progesterone concentrations during or immediately after treatment. The duration of the luteal phase in the non-pregnant bitches did not differ significantly from that in control dogs, as opposed to the premature cessation of the luteal phase when agle´pristone is administered in midgestation [11,13]. However, the intervals during which the plasma progesterone concentration exceeded 32 and 64 nmol/l were significantly shorter than in untreated bitches. The administration of agle´pristone during the early luteal phase does therefore affect progesterone production but not sufficiently to shorten the luteal phase. Shortening of the luteal phase in bitches after administration of agle´pristone in midgestation is associated with increased plasma levels of PGFM, the main metabolite of prostaglandin F2a, probably induced by fetal expulsion [11,16]. An increased level of prostaglandin F2a, could not have caused the effects on the progesterone secretion in the bitches of our study since they were not pregnant. Moreover, early termination of pregnancy would not lead to abortion but rather to resorption of the fetuses, which would not induce an increase in plasma PGFM concentrations [7,11]. It is also questionable whether in this part of the luteal phase prostaglandin F2a release could have caused premature luteolysis, since the rapidly growing cyclic and pregnant corpus luteum is relatively resistant to prostaglandin F2a [1,22,23]. Alternatively, the affected progesterone secretion in our bitches may be ascribed to the fact that occupation of the progesterone receptors by agle´pristone may have reduced the autoregulatory positive feedback of progesterone on its own secretion during the early luteal phase [24]. In the present study, administration of agle´pristone in the early luteal phase resulted in shortening of the interestrous interval. Since the duration of the luteal phase was unaffected, this can only be explained by an early termination of anestrus. In the bitch, increased release of GnRH by the hypothalamus [25] and increased sensitivity of the pituitary to GnRH from early to late anestrus [26] have been reported to be important determinants of the initiation of a new follicular phase. Furthermore, progression from early to late anestrus is associated with a rise in basal plasma FSH without a concomitant rise in basal LH [15]. The shortening of the interestrous interval by agle´pristone is presumably due to an effect on the hypothalamic–pituitary–ovarian axis. In contrast to the effects of agle´pristone when administered in midgestation [11,13], its administration in the early luteal phase in the cyclic dog did not induce a significant change in plasma prolactin concentration. Similar observations have been noted in early pregnancy termination [11]. Elevated plasma prolactin concentrations after administration of progesterone receptor antagonist in mid-gestation occur as a result of the occupation of the progesterone receptors by agle´pristone, which may mimic a sudden decline of plasma progesterone concentration and consequently induce enhanced prolactin release. This could be similar to the pathogenesis of pseudopregnancy, in which a rapid decrease in plasma progesterone concentration induces a rise in plasma prolactin concentration [4,12]. S. Galac et al. / Theriogenology 62 (2004) 494–500 499

The absence of a rise in prolactin after administration of agle´pristone in our study cannot be explained by the inability of the pituitary to increase prolactin secretion in this part of the luteal phase, for Okkens et al. [17] observed a significant increase in plasma prolactin concentration in bitches following hysterectomy at 8–16 days after ovulation, i.e., early in the luteal phase. However, the number of pituitary progesterone receptors may be lower in the early luteal phase than in midgestation. The expression of progesterone receptors in the uterus and ovary in the early luteal phase is similar to that in midgestation [9,27], but quantitation of progesterone receptors in the canine pituitary has not been reported. Another explanation for the absence of a rise in prolactin after administration of agle´pristone could be that early in gestation, when the embryo is very sensitive to changes in its environment, the action of the progesterone receptor antagonist is sufficient only to induce pregnancy termination but not to increase plasma prolactin concentration. Agle´pristone has been shown to be very effective in the early termination of pregnancy [11]. In cases of early pregnancy termination because of unwanted mating, agle´pristone is administered shortly after ovulation, when the diagnosis of pregnancy is not yet possible. Feldman et al. [8] reported that only 40% of bitches conceived after an unwanted mating, e.g., mating without fertility control. This implies that when agle´pristone is used as an abortifacient soon after mating, a large proportion of the dogs treated will not be pregnant. This study therefore used non-pregnant bitches to study the effects of agle´pristone administered during the early luteal phase. In conclusion, this study has shown that administration of agle´pristone in the first part of the luteal phase affects plasma progesterone concentration but not sufficiently to shorten the luteal phase. Plasma prolactin concentration was not affected. The interestrous interval in bitches treated with agle´pristone was shortened, suggesting that in the early luteal phase agle´pristone influences the hypothalamic–pituitary–ovarian axis.

Acknowledgements

The authors are grateful to Mrs. D.M. Blankensteijn for technical assistance. We thank Mr. S. Woitkowiak from Virbac Laboratories for the generous gift of Alizine1.

References

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TERMINATION OF MID-GESTATION PREGNANCY IN BITCHES WITH AGLEPRISTONE, A PROGESTERONE RECEPTOR ANTAGONIST

S. Galac,lv2 H.S. Kooistra,2a J. Butinar,’ M.M. Bever~,~ S.J. Dieleman: G. Voorhout4 and A.C. Okkens*

‘Small Animal Clinic, Veterinary Faculty, University of Ljubljana, Slovenia *Department of Clinical Sciencesof Companion Animals, 3Department of Farm Animal Health and 4Division of Diagnostic Imaging, Faculty of Veterinary Medicine, Utrecht University, The Netherlands

Received for publication: ~~~18, 1 ggg Accepted: August 4, 1999

ABSTRACT

Six pregnancies were terminated in mid-gestation with aglepristone, a progesterone receptor antagonist, in 5 beagle bitches in order to determine the effects of aglepristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase, and the interestrous interval. In addition, the effects of aglepristone on the condition of the uterus and fetuses were examined by ultrasonography. After confirmation of pregnancy by ultrasonography, the dogs received 10 mg, SC aglepristone per kg body weight on 2 consecutive days at about 30 d post ovulation. Before, during and after treatment with aglepristone, plasma samples were collected for determination of the concentrations of prolactin and progesterone. The condition of the uterus and fetuses was assessed by ultrasonography the day before and at least 3 times a week for at least 2 wk after aglepristone administration. Termination of pregnancy occurred within 4 to 7 d after the start of aglepristone treatment, which was well tolerated, with no side-effects except slight vaginal discharge. The results of ultrasonographic examination indicated that aglepristone leads to abortion but not to fetal resorption. Elevated plasma concentrations of prolactin were observed during aglepristone treatment, while plasma progesterone levels remained unchanged. Pregnancy termination with aglepristone resulted in premature cessation of luteal function. In addition, the interestrous interval was shortened. The latter effects may be the consequence of actions of the progesterone receptor antagonist at the hypothalamus-pituitary level. In conclusion, aglepristone proved to be a safe and effective abortifacient in mid-gestation in the bitch. The results of the present study also indicated that aglepristone directly or indirectly influences pituitary function. 0 2OlM by Elsevier Science Inc. Key words: abortion, progesterone receptor antagonist, prolactin, dogs

Acknowledgments: The authors thank Mrs. D.M. Blankenstein, Mr. A.V.P. van de Poll and Mrs. Y.W.E.A. Pollak for technical assistanceand Prof. Dr. A. Rijnberk, Dr. M. Kosec and Dr. B.E. Belshaw for critical reading of the manuscript. a Correspondence and reprint requests: [email protected]

Therfogenology 53:941-950.2000 0093-691 WOO/$-see front matter 0 2000 by Elsevier Science Inc. PII 50093-691 X(00)00241 -7 942 Theriogenology

INTRODUCTION

For many decades unwanted pregnancy in dogs has been avoided early in gestation by relatively large doses of estrogens (37). Administration of estrogens prolongs the oviductal transport time and tightens the utero-tubular junction, resulting in implantation failure and embryonic death (19). However, the use of estrogens can result in side-effects such as cystic endometrial hyperplasia (CEH), pyometra, and bone marrow suppression (l), although the occurrence of these side-effects was lower when relatively low doses of estradiol benzoate were administered on Days 3,5 and 7 after mating (41).

Maintenance of pregnancy in the bitch is dependent on ovarian secretion of progesterone by the corpus luteum (CL) throughout gestation (5,39,40). During the second part of the luteal phase, luteotropic factors originating from the pituitary such as prolactin and possibly luteinizing hormone are essential for the maintenance of the CL (7,25,26,32). Consequently, unwanted pregnancy in dogs can be terminated during mid-gestation by pharmacological agents which suppress prolactin release or interfere with the action or synthesis of progesterone. However, use of many of the currently available abortifacients is accompanied by unwanted and sometimes severe side-effects (2).

Repeated administration of prostaglandin Fza and its analogues during mid-term pregnancy in the bitch results in luteolysis (5,13). The narrow margin between a lethal dose (LD& and a therapeutic one; side-effects such as vomiting, diarrhea, hyperpnea and ataxia; and the need for repeated administration are important factors limiting the use of prostaglandins in veterinary practice (12,13). Dopamine agonists, such as bromocriptine and cabergoline, cause luteolysis by suppressing pituitary prolactin secretion. Reports on their efficacy differ, depending on the dose and the day of pregnancy at which administration is begun (l&29,46). High doses of bromocriptine frequently cause vomiting, nausea, lethargy and anorexia (7). Cabergoline has been reported to be better tolerated (32,35). The combined use of cabergoline and prostaglandins has also been described (30). When used in combination, they are generally effective at lower doses than with single administration and result in fewer side- effects (3 1).

Because of the undesirable side-effects of the above mentioned drugs, attention has been given to agents that prevent the action of progesterone, i.e., progesterone receptor antagonists. Mifepristone (RU 486) has proven to be an effective abortifacient in the dog (8,24,42), but is not available for clinical use. Recently, another progesterone receptor antagonist, aglepristone, has been marketed in France as an abortifacient for veterinary use. Administration of aglepristone effectively terminated pregnancy in 66 of 69 bitches in which pregnancy had been confirmed by ultrasonography (14).

The purpose of the present study was to determine the effects of aglepristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase and the interestrous interval in bitches in which it was given to terminate pregnancy at mid-gestation. In addition, the effects of aglepristone on the condition of the uterus and fetuses were examined by ultrasonography. Theriogenology 943

MATERIALS AND METHODS

Animals, Treatment and Collection of Blood Samples

Five clinically healthy beagle bitches, 4 to 6 yr of age, were housed singly or in pairs in indoor-outdoor runs, fed a standard commercial dry dog food once daily, and given water ad libitum. The dogs were examined 3 times a week for the presence of vulvar swelling and serosanguinous vaginal discharge, which were considered to signify the onset of proestrus. The day on which the plasma progesterone concentration reached 5 pg/L for the first time after the onset of proestrus was defined as the day of ovulation, i.e., the first day of the luteal phase (Day 1). The luteal phase was considered to be terminated when the plasma concentration of progesterone fell below 1 pg/L for the first time. The interestrous interval was defined as the period from 1 ovulation to the next. All beagle bitches were mated 1 or 2 d after the estimated day of ovulation, and all became pregnant. One bitch was mated and became pregnant during 2 successive estrous cycles. Pregnancy was confirmed by ultrasonographic examination at about Day 28. After confirmation of pregnancy the dogs were treated subcutaneously with aglCpristone (Alizine,R Hoechst Roussel Vet, Romainville, France) on 2 consecutive days at a dose of 10 mg/kg body weight, once a day, starting approximately 30 days after ovulation. Following the administration of aglCpristone, ultrasonography was performed 3 times a week to assess the condition of the fetuses by determining cardiac activity. In addition, the architecture of the uterus was studied for at least 2 wk. Pregnancy was considered to be terminated on the day on which fetuses could no longer be detected in the uterus. All ultrasonographic examinations were performed through the ventral abdominal wall, with the dogs in dorsal recumbency, using a high definition ultrasound system (HDI 3000, Advanced Technology Laboratories, Woerden, The Netherlands) equipped with a 10-5 MHz broadband linear array transducer. Plasma concentrations of progesterone and prolactin were determined 3 times per week from the start of proestrus until the end of the luteal phase. Additionally, on the days of administration of aglkpristone the plasma concentrations of progesterone and prolactin were determined immediately before and at 1, 2, 4 and 8 h after treatment. During the 10 d after administration of aglkpristone plasma concentrations of progesterone and prolactin were determined once daily. Blood samples were collected by jugular venipuncture into evacuated heparinized tubes. The samples were centrifuged, and plasma was stored at -20 ‘C until levels of progesterone and prolactin were analyzed.

Radioimmunoassays

Concentrations of progesterone in the peripheral blood were measured by a previously validated radioimmunoassay (9,27). The intra-assay and inter-assay coefficients of variation were 11 and la%, respectively, and the limit of detection was 0.1 pg/L.

Concentrations of prolactin in the peripheral blood were determined by a previously validated heterologous radioimmunoassay (27). The intra-assay and inter-assay coefficients of 944 Theriogenology

variation were 3.5 and 11 S%, respectively. The lowest detectable concentration of prolactin was 0.8 pg/L.

Statistical Analysis

Plasma prolactin concentrations 2 50 pg/L were considered to be 50 @L. For each dog mean plasma prolactin concentrations before treatment, during treatment and after treatment (Day 36 until the end of the luteal phase) were compared by an ANOVA for repeated measures. Subsequently, multiple comparisons were performed using the Student-Newman- Keuls test. The mean duration of the luteal phase and mean interestrous interval in the dogs treated with aglepristone were compared with these same variables in untreated beagle dogs in our colony using the Student’s t-test (two-tailed). Values are expressed as a range or as mean + SEM. Values were considered to be significantly different at P 5 0.05.

RESULTS

Pregnancy was confirmed in all dogs at 27 to 31 d following mating. The fetuses were alive and thenumber ranged from 2 to 6 (mean 4.5). The bitch that was mated during 2 successive estrous cycles conceived normally with 5 fetuses the first time and 6 fetuses the second time.

Aglepristone treatment was well tolerated and no side-effects were observed except for a small amount of mucoid vaginal discharge during the week after the start of treatment. Two days after the start of the administration of aglepristone both living and dead fetuses were observed in 2 dogs, while in the remaining dogs all the fetuses were alive. One to two days later all the fetuses in 5 dogs were dead, with whole fetuses or remnants of fetuses visible in 3 of the dogs. In 1 of these 3 dogs contractions of the uterine wall were observed, with expulsion of a dead fetus from the placentation site. In the remainingdogsall fetuses were still alive. Again, 3 to 4 d later no fetuses or remnants of fetuses were observed anymore.

Ovoid placentation sites of the uterus were broader than the interplacentation zones in all the dogs and had echogenic luminal contents with very poor distinction between wall and contents. During the next 6 to 11 d the placentation sites became smaller but were still present in all the dogs 14 to 18 d following the administration of aglepristone. In one 5-yr-old bitch vaginal discharge was observed 4 wk after initiation of the aglepristone treatment. Ultrasonographic examination of the uterus at the same time revealed cystic and hyperplastic changes compatible with cystic endometrial hyperplasia (CEH).

Plasma progesterone levels did not change during treatment with aglepristone, and all pregnancies were terminated despite the presence of relatively high plasma concentrations of progesterone. However, after treatment, plasma progesterone concentrations declined to less than 1 pg/L within 8 to 34 d (mean 19 f 4 d). Thus, the mean duration of the luteal phase in which the beagIes were treated with aglepristone (52+4 d) was significantly shorter (P=O.OOS) than that of untreated beagle dogs (75 f 4 d; ref. 27). The interestrous interval in which the Theriogenology 945

beagles were treated with aglCpristone (1X* 10 d) was significantly (P~0.05) shorter than that in another group of 6 beagle bitches in our dog colony (199k 15 d).

Plasma prolactin concentrations were clearly elevated within 24 h after the start of the treatment with aglbpristone and returned to basal levels in 2 to 4 d (Figure 1). Mean plasma prolactin concentrations in all bitches during treatment (range: 26.4 to 47 @L) were significantly higher (PcO.001) than those before (range: 3.3 to 13.8 pg/L) and after (range: 2.7 to 13.2 pg/L) treatment (Figure 2). However, high plasma prolactin concentrations (>lO cl&) were also occasionally observed before and after treatment with aglCpristone.

Days Figure 1. Plasma concentrations of prolactin (0) and progesterone (0) in a 4-yr-old beagle bitch, starting from the day of ovulation (Day 1) to the end of the luteal phase. On Days 30 and 3 1, the bitch was treated subcutaneously with aglCpristone at a dose of 10 mgIkg body weight, once each day.

50 - *** ***

40 - I-t::3 30 - ‘, I. .:., ,

20 -

10 - ,_ -r ,2,/I ,:“. l- ,, ‘/ .I,,,_ I ., ,_,, ,,,; 0 :, ,, , I Before During After Figure 2. The average (+ SEM) of the mean plasma prolactin concentrations in 6 beagle bitches before, during and after treatment with aglCpristone. Asterisks indicate significant difference. 946 Theriogenology

DISCUSSION

The results of this study demonstrate that aglepristone, a new progesterone receptor antagonist, is a safe and effective abortifacient for use during mid-gestation in the bitch. All pregnancies were successfully terminated 4 to 7 d after initiation of treatment with aglepristone. Rapid and effective termination of pregnancies is consistent with the results of FiCni et al. (14) who employed aglepristone, and of Concannon et al. (8) and Linde-Forsberg et al. (24) who used mifepristone. In humans, combined use of progesterone receptor antagonists with prostaglandins has proven to be more effective than the use of progesterone receptor antagonists alone (4). Taking into account the results of the present study and the reported high effectiveness rate of aglepristone (>95%) in terminating pregnancy during mid- gestation as well as the side-effects of prostaglandins (12,13), the combination of aglepristone and prostaglandins is not recommended in dogs.

Aglepristone can be used effectively as an alternative to estrogens to avoid unwanted pregnancy early in gestation in the bitch (14). In our study we used only bitches with confirmed pregnancy. It may be advisable to use aglepristone only after pregnancy is confirmed, since Feldman et al. (12) reported that only 40% of the bitches conceived after an unwanted mating (i.e., mating without fertility control). When mating occurs within a fertility program a pregnancy rate higher than 90% can be expected (45).

The appearance of the uterus following the disappearance of whole fetuses or of remnants of fetuses resembles that of a postpartum uterus, as described by Pharr and Post (34), and differs from that of a uterus following fetal resorption (11). This appearance together with the observation of contractions of the uterine wall with the expulsion of a dead fetus from the placentation site indicate that the administration of aglepristone leads to abortion and not to fetal resorption.

The only side-effect of aglepristone treatment observed in this study was some mucoid vaginal discharge in the first week after administration. In one 5-yr-old bitch vaginal discharge was also observed 4 wk after the start of treatment with aglepristone. In this dog cystic and hyperplastic changes compatible with CEH were detected by ultrasonography of the uterus. Considering the central role of progesterone in the pathogenesis of CEH (10,22,43) and the relatively long interval between treatment with aglepristone and ultrasonographic detection of CEH, the latter should probably not be regarded as a side-effect of aglepristone treatment. However, the possibility that aglepristone treatment might reinforce the development of an already existing mild case of CEH can not be excluded.

One bitch was mated twice and became pregnant with 6 fetuses after having been treated with agiepristone during the previous pregnancy. Similar results have been reported in studies on RU 486 (23,24). These observations suggest that progesterone receptor antagonists do not impair future fertility, an important prerequisite for an abortifacient.

The still high plasma progesterone concentrations at the time of fetal death indicate that pregnancy was not terminated by a decline in plasma progesterone concentrations, as Theriogenology 947

suggested by Concannon and Hansel (S), but probably by a direct effect of aglepristone at the level of the uterine progesterone receptor (824). Following binding to the progesterone receptor, a progesterone receptor antagonist stabilizes the receptor’s structure by its high affinity and prevents progesterone from exerting its biological effect (8,44)

Termination of pregnancy with aglepristone resulted in premature cessation of luteal function. Luteolysis secondary to abortion or resorption can be related to uterine release of luteolytic amounts of prostaglandin FZa (8). Similar to this findings administration of prostaglandin Fza to induce abortion in bitches is also associated with a shortened luteal phase (36). Pregnancy termination by the progesterone receptor antagonist mifepristone (RU 486) causes increasesin peripheral concentrations of PGFM, the main metabolite of prostaglandin Fza (24). Such an increase can also be expected to occur when aglepristone is used as an abortifacient. Studies of RU 486 in humans have suggested another possibility for the early cessation of the luteal phase. Based on the observation that there is a decrease in LH pulse amplitude, LH pulse frequency and LH secretion in humans during treatment with RU 486, it has been speculated that premature luteolysis may be an effect of the progesterone receptor antagonist at the hypothalamic-pituitary level (15,38). Detailed studies in dogs on the effect of progesterone receptor antagonists on the hypothalamic-pituitary-ovarian axis are lacking. However, the observed shortening of the interestrous interval associated with aglepristone treatment also suggests an influence on the hypothalamic-pituitary-ovarian axis. The aglepristone-induced shortening of the interestrous interval in the bitches in this study cannot be explained by the shortening of the luteal phase alone and thus may be due to shortening of anestrus. Progression from early to late anestrus in the bitch is associated with an increase in circulating FSH without a concomitant rise in basal LH concentration (2 1). This indicates that an increase in circulating FSH is a critical event required for the initiation of ovarian folliculogenesis and consequently for the termination of anestrus. Therefore, it may be hypothesized that the aglepristone-induced shortening of the interestrous interval is directly or indirectly the result of enhanced pituitary FSH release.

There are also other indications that aglepristone has central effects in the bitch. Treatment with aglepristone resulted in a pronounced increase in plasma prolactin concentrations within 24 h. Elevated plasma prolactin concentrations have also been reported in bitches with overt pseudopregnancy (28). A rapid decline in the plasma concentration of progesterone may be a precipitating factor in the pathogenesis of pseudopregnancy (16,17). Correspondingly, ovariectomy performed in the luteal phase of the estrous cycle often induces overt pseudopregnancy. The rapid decline in plasma concentrations of progesterone is assumed to cause prolactin release, which in turn would give rise to pseudopregnancy. In addition, at the time of parturition plasma prolactin levels in the bitch rise in response to the sharp decline in the plasma progesterone concentration (6). Occupation of the central progesterone receptors by the antagonist aglepristone may mimic a sudden decline in progesterone concentration and consequently result in increased prolactin release. According to our findings, progesterone receptor antagonist-induced elevation of plasma prolactin values cannot be explained by intrauterine fetal death, as proposed by Linde-Forsberg et al. (24), because fetal death occurred at least 2 d after the rise in plasma prolactin concentration. 948 Theriogenology

High plasma prolactin concentrations were also observed occasionally before and after treatment with aglkpristone. Studies in which plasma prolactin concentration was measured daily have also demonstrated such peak values (6,27,33). This might indicate pulsatile secretion of prolactin in the bitch, as has been found for several other pituitary hormones (3,20,21). However, studies to determine whether the secretion of prolactin is pulsatile in the bitch have not yet been performed.

In conclusion, aglepristone proved to be a safe, reliable and effective abortifacient during mid-gestation in the bitch. AglCpristone treatment was associated with a shortening of the luteal phase and the interestrous interval, and resulted in temporarily highly elevated plasma prolactin levels.

REFERENCES

1. Bowen RA, Olson PN, Behrendt MC, Wheeler SL, Husted PW, Nett TM. Efficacy and toxicity of estrogens commonly used to terminate canine pregnancy. J Am Vet Med Assoc 198.5;186:783-788. 2. Braakman A, Okkens AC, Van Haaften B. Medical methods to terminate pregnancy in the dog. Comp Contin Educ Pratt Vet Small Anim Pratt 1993;15:1505-1512. 3. Brabant G, Prank K, SchGfl C. Pulsatile patterns in hormone secretion. Trends Endocrinol Metab 1992;3:183-190. 4. Bygdeman M, Swahn M-L. Progesterone receptor blockage. Effect on uterine contractility and early pregnancy. Contraception 1985;32:45-52. 5. Concannon PW, Hansel W. Prostaglandin Fzcc induce luteolysis, hypothermia and abortion in beagle bitches. Prostaglandins 1977;13:533-542. 6. Concannon PW. McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil 1989;39(Suppl):3-25. 7. Concannon PW, Weinstein R, Whaley S, Frank D. Suppression of luteal function in dogs by luteinizing hormone antiserum and bromocriptine. J Reprod Fertil 1987;8 1: 175- 180. 8. Concannon PW, Yaeger A, Frank D, Iyampillai A. Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs. J Reprod Fertil 1990;88:99-104. 9. Dieleman SJ, Schoenmakers HJN. Radioimmunoassays to determine the presence of progesterone and estrone in the starfish Asterias rubens. Gen Comp Endocrinol 1979;39:534-542. 10. Dow C. The cystic hyperplasia-pyometra complex in the bitch. Vet Ret 1958;70: 1102- 1110. 11. England GCW. Ultrasonographic assessment of abnormal pregnancy. Vet Clin North Am (Small Anim Pratt) 1998;28:849-868. 12. Feldman EC, Davidson AP, Nelson RW, Nyland TG, Munro C. Prostaglandin induction of abortion in pregnant bitches after misalliance. J Am Vet Med Assoc 1993:202;1855- 1858. 13. FiCni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for pregnancy termination in bitches using prostaglandin Fza, J Reprod Fertil 1997;51(Suppl):245-250. Theriogenology 949

14. FiCni F, Tainturier D, Bruyas JF, Badinand F, Berthelot X, Ronsin P, Rachail M, Lefay MP. Etude clinique d‘une anti-hormone pour provoquer l’hvortement chez la chienne: I’aglCpristone. Ret Med Vet 1996;172:359-367. 15. Garzo VG, Liu J, Ulmann .A, Baulieu EE, Yen SSC. Effects of an antiprogesterone (RU 486) on the hypothalamic-hypophyseal-ovarian-endometrial axis during the luteal phase of the menstrual cycle. J Clin Endocrinol Metab 1988;66:508-517. 16. Gerres S, Hijveler R, Evers P, Hoffmann B. Investigations on the role of progesterone (P4) in the endocrine control of overt pseudopregnancy in the bitch, application of an antigestagen. Proc 1 lth Int Congr Anim Reprod Artif Insem 1988;90-91. 17. Harvey MJA, Dale MJ, Lindley S, Waterston MM. A study on the aetiology of pseudopregnancy in the bitch and the effect of cabergoline therapy. Vet Ret 1999;144:433-436. 18. Jiichle W, Arbeiter K, Post K, Ballabio R, D’Ver ASD. Effects on pseudopregnancy, pregnancy and interoestrous intervals of pharmacological suppression of prolactin secretion in female dogs and cats. J Reprod Fertil 1989;39(Suppl): 199-207. 19. Jiichle W, Lamond DR, Andersen AC. Mestranol as an abortifacient in a bitch. Theriogenology 1975;4: l-9. 20. Kooistra HS, Greven SH, Mol JA, Rijnberk A. Pulsatile secretion of a-MSH and the differential effects of dexamethasone and haloperidol on the secretion of c(-MSH and ACTH in dogs. J Endocrinol 1997;152:113-121. 21. Kooistra HS, Okkens AC, Bevers MM, Popp-Snijders C, Van Haaften B, Dieleman SJ, Schoemaker J. Concurrent pulsatile secretion of luteinizing hormone and follicle- stimulating hormone during different phases of the estrous cycle and anestrus in beagle bitches. Biol Reprod 1999,60:65-71. 22. Kooistra HS, Okkens AC, Mol JA, Van Garderen E, Kirpensteijn J, Rijnberk A. Lack of association of progestin-induced cystic endometrial hyperplasia with GH gene expression in the canine uterus. J Reprod Fertil 1997;51(Suppl):355-361. 23. Lavaud J. Emploi d‘un antagoniste de la progesdrone, le RU 38346, abortif chez la chienne. Reproduction 1989;24:253-258. 24. Linde-Forsberg C, Kindahl H, Madej A. Termination of mid-term pregnancy in the dog with oral RU 486. J Small Anim Pratt 1992;33:33 l-336. 25. Okkens AC, Bevers MM, Dieleman SJ, Willemse AH. Evidence for prolactin as the main luteotrophic factor in the cyclic dog. Vet Quart 1990;12: 193-201. 26. Okkens AC, Dieleman SJ, Bevers MM, Lubberink AAME, Willemse AH. Influence of hypophysectomy on the lifespan of the corpus luteum in the cyclic dog. J Reprod Fertil 1986;77:187-192. 27. Okkens AC, Dieleman SJ, Bevers MM, Willemse AH. Evidence for the non-involvement of the uterus in the lifespan of the corpus luteum in the cyclic dog. Vet Quart 1985;7: 169- 173. 28. Okkens AC, Dieleman SJ, Kooistra HS, Bevers MM. Plasma concentrations of prolactin in overtly pseudopregnant Afghan hounds and the effect of metergoline. J Reprod Fertil 1997;51(Suppl):295-301. 29. Onclin K, Silva LDM, Donnay I, Verstegen JP. Luteotrophic action of prolactin in dogs and the effects of a dopamine agonist, cabergoline. J Reprod Fertil 1993:47(Supp1);403- 409. 950 Theriogenology

30. Onclin K, Silva LDM, Verstegen JP. Termination of unwanted pregnancy in dogs with the dopamine agonist, cabergoline, in combination with a synthetic analogue of PGFZalpha, either cloprostenol or alphaprostol. Theriogenology 1995;43:813-822. 3 1. Onclin K, Verstegen JP. Practical use of a combination of a dopamine agonist and a synthetic prostaglandin analogue to terminate unwanted pregnancy in dogs. J Small Anim Pratt 1996;37:21 l-216. 32. Onclin K, Verstegen JP. In vivo investigation of luteal function in dogs: effects of cabergoline, a dopamine agonist, and prolactin on progesterone secretion during mid- pregnancy and -diestrus. Dom Anim Endocrinol 1997: 14;25-38. 33. Onclin K, Verstegen JP. Secretion patterns of plasma prolactin and progesterone in pregnant compared with nonpregnant dioestrous beagle bitches. J Reprod Fertil 1997;51(Suppl):203-208. 34. Pharr JW, Post K. Ultrasonography and radiography of the canine postpartum uterus. Vet Radio1 Ultrasound 1992;33:35-40. 35. Post K, Evans LE, Jochle W. Effects of prolactin suppression with cabergoline on the pregnancy of the bitch. Theriogenology 1988:29;1233-1243. 36. Romagnoli SE, Camillo F, Cela M, Johnston SD, Grassi F, Ferdeghini M, Aria G. Clinical use of prostaglandin FzU to induce early abortion in bitches: serum progesterone, treatment outcome and interval to subsequent oestrus. J Reprod Fertil 1993:47(Suppl): 425-431. 37. Rowlands IW. Anterior pituitary hormones and the oestrus cycle. Vet Ret 1948;60:181- 186. 38. Schaison G, George M, LeStrat N, Reinberg A, Baulieu EE. Effects of the antiprogesterone steroid RU 486 during midluteal phase in normal women. J Clin Endocrinol Metab 1985;61:484-489. 39. Sokolowski JH. The effects of ovariectomy on pregnancy maintenance in the bitch. Lab Anim Sci 1971;21:696-699. 40. Sokolowski JH, Geng S. Effect of prostaglandin Fra THAM in the bitch. J Am Vet Med Assoc 1977;170:536-537. 41. Sutton DJ, Geary MR, Bergman JGHE. Prevention of pregnancy in bitches following unwanted mating: a clinical trial using low dose oestradiol benzoate. J Reprod Fertil 1997;5l(Suppl):239-243. 42. Taverne MAM, Van Der Weijden GC, Van Oord HA. Pregnancy and parturition in dogs: Approached from some diagnostic, patho-physiological and therapeutic points of view. In: Christiansen LJ (ed), Proc Symposium on Reproduction in the Dog, 1989:71-88. 43. Teunissen GHB. The development of endometritis in the dog and the effect of oestradiol and progesterone in the uterus. Acta Endocrinol 1952;9:407-420. 44. Tuyttens FAM, Macdonald DW. Fertility control: an option for non-lethal control of wild carnivores? Anim Welfare 1998;7:339-364. 45. Van Haaften B, Dieleman SJ, Okkens AC, Willemse AH. Timing the mating of dogs on the basis of blood progesterone concentration. Vet Ret 1989;125:524-526. 46. Wichtel JJ, Whitacre MD, Yates DJ, Van Camp SD. Comparison of the effects of PGFlu and bromocryptine in pregnant beagle bitches. Theriogenology 1990:33;829-836. PAPER Safety and efﬁcacy of mid-term pregnancy termination using aglepristone in dogs

OBJECTIVES: To investigate effects and side effects of aglepristone in The purpose of this study was to inves- terminating pregnancy in bitches. tigate the effects and side effects of the progesterone receptor antagonist aglepristone METHODS: Twenty-two bitches were treated in mid-pregnancy with in bitches in mid-pregnancy. subcutaneous injections of aglepristone at a total dose of 20 mg/kg. Short-term follow-up (one to two weeks after treatment) included MATERIALS AND METHODS clinical examination and abdominal ultrasonography in 18 of the dogs. Long-term telephone follow-up was recorded for all 22 dogs. Medical reports at Albano Animal Hospi- tal in Stockholm, Sweden from June 2002 RESULTS: Pregnancy was terminated in 21 bitches (95 per cent). to June 2006 were examined. Abdominal Signs of abortion occurred one to eight days after treatment. Vaginal ultrasound was performed on 82 bitches between day 23 and day 42 (median 30 discharge was evident in 17 (77 per cent) dogs. Obvious signs of days) after suspected mismating using a 5 parturition were seen in nine (41 per cent) dogs. Eight dogs (36 per to 12 MHz transducer (HDXE11; Philips). Twenty-eight (34 per cent) dogs were cent) developed anorexia, and in two (9 per cent) of the dogs a local judged as pregnant on ultrasonographic reaction at the injection site was evident. Two dogs developed examination. Nineteen (51 per cent) of a total of 37 dogs, in which mating had pyometra two and four years after treatment, respectively. been observed, were pregnant. Twenty- CLINICAL SIGNIFICANCE: Aglepristone, when administered in mid- two bitches were treated with the injectable oil-based solution aglepristone (Alizin; gestation, is effective in terminating pregnancy. Side effects are few Virbac). and transient. About 20 mg/kg aglepristone was the total dose. In 19 of the 22 dogs, the dose was divided and given on two consecutive days according to the label. Three of the C. H. PETTERSSON AND A. TIDHOLM INTRODUCTION dogs were given the total dose on a single occasion. A 1Á2 Â 40 mm (18G Â 1Á5 inch) Journal of Small Animal Practice (2009) 50, 120–123 Previously used medical methods of termi- needle was used. The subcutaneous in- DOI: 10.1111/j.1748-5827.2008.00692.x nating pregnancies in bitches include the jection site in the neck was massaged imme- use of oestrogens, prostaglandins, dopamine diately after the injection. Doses exceeding agonistsandglucocorticoids.Progesteroneis 10 ml were divided between two different a natural steroid hormone secreted by the injection sites. corpus luteum in the ovary. It plays a crucial A follow-up examination was per- role in establishing and maintaining preg- formed, in 18 of the 22 bitches, one to nancyinthebitch,asinotherspecies.Proges- two weeks after treatment with aglepri- terone has four major effects on maintaining stone. Three owners were interviewed by pregnancy (1) permitting the maturation of telephone at this time and one owner uterine cells to prepare them for implanta- was interviewed one month after treat- tion, (2) stimulating the glandular secretions ment. The examination included inspec- of the endometrium required for embryo tion of the injection site and the vulva development, (3) closing the cervix and (4) for any visible vulvar discharge. The uterus reducing uterine contractility. Interrupting was examined by ultrasound, and diameter or interfering with the supportive action of of uterine horns measured. All owners progesterone on the uterus is one means of completed a questionnaire, including ques- terminating pregnancy (Concannon and tions on the presence of vulval discharge, others 1990, Sankai and others 1991, loss of appetite, vomiting, diarrhoea or Albano Animal Hospital, Rinkebyva¨gen 23, 182 Linde-Forsberg and others 1992, Galac other abnormalities, and the timing and 36 Danderyd, Sweden and others 2000, Fieni and others 2001). duration of these signs.

120 Journal of Small Animal Practice Á Vol 50 Á March 2009 Á Ó 2009 British Small Animal Veterinary Association Aglepristone and mid-term pregnancy

All 22 owners were interviewed by tele- Table 1. Ultrasonographic evaluation of the uterus at follow-up examination phone during October 2007 (1Á3to5Á3 Treatment day after mismating Bodyweight Day(s) of Uterine diameter years after treatment) using a questionnaire (kg) follow-up after at follow-up (cm) that included the subsequent questions (1) treatment the present health of the bitch, (2) the tim- 23 4 13 Not visible ing of ﬁrst oestrus after aglepristone treat- 25 9Á5 7 Not visible ment and intervals between the following 26 22Á5111Á25-1Á45 28 21 13 Not visible oestrus cycles, (3) whether the bitch had 28 6Á8100Á5 undergone ovariohysterectomy, the reason 28 4Á87 1 for this and the timing after treatment, (4) 28 9 8 1 28 7Á4 6 and 12 2 and 1 whether the bitch had been successfully 28 10Á67 1Á8-2Á2 bred after the aglepristone treatment and 30 29 15 0Á5 (5) whether or not the owner was satisﬁed 30 35Á49 0Á5 30 3 13 0Á48-0Á87 with the treatment of mismating. 30 47 10 1 30 7Á1101 Statistical methods 30 27 7 1Á3 30 5Á4121Á45 Statistical calculations were performed by 30 35 14 1Á45 use of a computerised statistical program 30 3Á4 16 Not measured* (JMP 3.2; SAS Institute Inc.). Data are pre- 28 37 y 28 37 y sented as median and range. 42 29 y 41 15 y

*Viable foetuses RESULTS yNo ultrasound follow-up

The 22 dogs ranged in age from nine to 101 months (median 43 months). The dogs dogs, the discharge was detected from one fever, vaginal discharge and an enlarged weighed between 3 and 47 kg (median to two days after treatment. In one dog, the uterus (1 to 2 cm in diameter). Although 12Á8 kg). Six (27 per cent) of the dogs were discharge was minimal, brownish and evi- no sample of the vaginal discharge was sub- mixed breeds, two (9 per cent) were papil- dent six to 11 days after treatment. In most mitted for bacterial culture, this dog was lons, two were American cocker spaniels of the dogs, the discharge was still visible at prescribed enrofloxacin (Baytril Vet; Bayer and two were cavalier King Charles spaniels. the time of the first follow-up examination. Healthcare AB) at a dose of 5 mg/kg There was one (5 per cent) of each of the Signs of parturition, such as shivering, bodyweight once daily orally for 10 days. following breeds: shih-tzu, Groenendael, panting or nesting behaviour, were seen At re-examination six days later, the body Lo¨wchen, Tibetan spaniel, Irish red and in nine of the 22 (41 per cent) dogs. These temperature had normalised and the diam- white setter, Terveuren, Estrela mountain clinical signs were detected over one to two eter of the uterus measured less than 1 cm. dog, Shetland sheepdog, Slovensky kopov days from day 1 or 2 after treatment, except The bitch was considered clinically healthy. and rottweiler. in one dog where the signs lasted for seven Long-term follow-up was performed, Pregnancy was terminated in 21 bitches days (from day 2). The dog, in which the between 1Á3 and 5Á3 years after treatment, (95 per cent). Seventeen of the 22 dogs (77 treatment failed showed signs of parturi- by telephone using a questionnaire. One per cent) were treated between day 28 and tion but had no visible vaginal discharge. (5 per cent) of the dogs was euthanased day 30. In two of these dogs, the owners Eight (36 per cent) of the dogs were six months after treatment because of pre- detected parts of foetuses in the discharge. anorexic for several days (four to 10 days). existing epilepsy. The remaining 21 (95 per Three of the remaining dogs were treated One of the dogs lost 3 kg (10 per cent of its cent) dogs were considered healthy. One of between day 23 and day 26, and in these bodyweight) within 14 days after treat- the 22 dogs underwent ovariohysterectomy dogs no vaginal discharge was detected. ment. One dog (5 per cent) had polydipsia because of treatment failure. Seventeen of An abortion with noticeable foetuses for five days from one day after treatment. the remaining 20 (85 per cent) bitches occurred in two dogs treated on day 41 Two dogs (9 per cent) developed a local (including the bitch that was treated and day 42 after mating, respectively. reaction with swelling at the injection site. because of a suspected endometritis) were, Eighteen of the 22 treated dogs were One of these reactions was detected by the according to the owners, still cycling with evaluated by ultrasound seven to 16 days owner after seven days and disappeared normal oestrus at the time of follow-up. after treatment. The size of the remnants afteracoupleofdays.Intheotherdog,asoft Ten (50 per cent) of these bitches had a nor- of the embryonic cavities varied from unde- fluctuating swelling was detected by the mal interoestrus interval, three (15 per tectable to 2Á2 cm in diameter (Table 1). veterinarian at the time of the follow-up cent) a prolonged interval and two (10 Viablefoetuses were seen in one (5per cent) examination. per cent) a shortened oestrus interval the dog. Vaginal discharge was observed in 17 One (5 per cent) dog had a suspected first oestrus after the treatment, according of the 22 (77 per cent) dogs. In 16 of these endometritis six days after treatment, with to their owners. In the remaining five dogs,

Journal of Small Animal Practice Á Vol 50 Á March 2009 Á Ó 2009 British Small Animal Veterinary Association 121 C. H. Pettersson & A. Tidholm

no information was available concerning that diethylstilbestrol had little effect It is interesting to note that only 19 of 37 time of first oestrus cycle after treatment. although oestradiol cypronate had a better dogs (51 per cent) in which mating had Two bitches developed pyometra and efficacy. Oestrogens used in dioestrus have been observed were pregnant. This indi- underwent ovariohysterectomy, two and been shown to result in high incidence of cates that early use of pharmaceutical four years after treatment, at nine and seven pyometra (Bowen and others 1985). A field agents to terminate or prevent pregnancy years old, respectively. In one dog, the investigation using oestradiol benzoate at may be unwarranted in many cases. Own- owner had elected for ovariohysterectomy low doses showed this treatment to be effec- ers should be informed of these facts and because of the presence of a male dog in the tive and resulted in a lower incidence of have the option of choosing what treatment household. In conclusion, four of 22 pyometra after treatment (Sutton and may be most suitable for their bitch. (18 per cent) bitches underwent ovario- others 1997). A recent study (Whitehead In accordance with this study, other hysterectomy, one because of treatment 2008) showed that incidence of pyometra studies reported effective abortion in at failure, one because of having a male was higher in dogs treated for mismating least 95 per cent of bitches treated in dog in the family and two because of devel- with low doses of oestradiol benzoate than mid-pregnancy (Galac and others 2000, oping pyometra. Four of 20 (20 per cent) in untreated bitches. Prostaglandins have Fieni and others 2001). It has been recom- dogs, excluding the dog that was eutha- effects on many different organs and have mended that the total dose of aglepristone nased because of epilepsy and the dog also been used to induce abortion. Their be divided into two injections given on ovariohysterectomised because of treat- efficacy as an abortive drug results from consecutive days. Three of the 22 dogs were ment failure, were bred after the treatment induction of uterine contractions and given the total dose as a single injection with aglepristone. Three bitches conceived luteolysis (Feldman and others 1993). because of problems with owner compli- when bred and had normal litters. One of However, prostaglandins also have many ance. The only bitch which did not abort, them was bred twice, one and two years side effects such as hyperventilation, vom- was one of these three dogs. It is possible after treatment and had two normal litters. iting, defecation, urination and pad sweat- that the use of a single dose caused treat- A second dog gave birth to one normal lit- ing. Natural prostaglandins must be given ment failure in this case. ter one year after treatment. The third dog two or three times daily for four to seven Side effects previously reported for agle- had two litters (two and three years after days. Synthetic prostaglandins have fewer pristone included local pain and occasional treatment). One dog did not conceive side effects and a longer duration of action, swelling at the injection site, vaginal dis- when bred at the first oestrus after treat- but increase the risk of death because of charge, discomfort, depression, anorexia ment, but later had two normal litters inadvertent overdose. Hospitalisation is and a shortened inter-oestrus interval at (one and three years after treatment). advised for the duration of treatment on first oestrus after the treatment (Galac The owners of all the bitches in which account of side effects and the necessity and others 2000, Hubler and Arnold the treatment was successful said that they of frequent injections (Fieni and others 2000). In the present study, local reactions were pleased with the method used to ter- 1989, Fieni and others 1997). were seen in two (9 per cent) of 22 dogs. minate pregnancy. The corpus luteum in the bitch is depen- Behavioural changes, such as discomfort dent on prolactin to maintain progesterone and depression, are expected in conjunc- levels during late pregnancy. Treatment tion with parturition. Anorexia was seen DISCUSSION with dopamine agonists that exert antipro- in eight (36 per cent) dogs. This has also lactinergic effect via stimulation of D2 been observed in other studies when the In the present retrospective study, aglepri- pituitary receptors or inhibition of central treatment was initiated in early pregnancy stone was shown to be effective at terminat- serotoninergic receptors from day 35 to 40 (Hubler and Arnold 2000, Galac and ing pregnancy in mid-gestation in dogs can result in abortion (Concannon and others 2004). with few and reversible side effects. Agle- others 1987, Post and others 1988, Onclin The size of the uterus after treatment dif- pristone, as is the case with some other and others 1993). There are also other fered between dogs treated at the same stage drugs mentioned below, can be used when methods of terminating pregnancy such of gestation (Table 1) and did not correlate pregnancy has been confirmed. as combining prostaglandins and dopa- with bodyweight. The differences might be Drugs used to terminate or prevent mine agonists, studies of which have shown because of the fact that the examination was pregnancy vary in effectiveness and magni- good results and fewer side effects (Onclin performed on different days (days 6–15) tude of side effects. Oestradiol benzoate is and others 1993, Onclin and others 1995, after treatment and by different veterinary licensed to prevent pregnancy. Oestrogen Onclin and Verstegen 1996, Gobello and surgeons. has a toxic effect on the oocytes, alters others 2002). Dexamethasone has also In conclusion, aglepristone is an effec- the transportation time in the oviducts been used to terminate pregnancy in tive drug for pregnancy termination, when and tightens the uterotubular junction. bitches (Zone and others 1995, Wanke used in mid-pregnancy, and is reasonably This leads to implantation failure and and others 1997). However, the use of well tolerated by dogs. Although treatment embryonic death (Jo¨chle and others corticosteroids to induce abortion is un- with aglepristone does not seem to nega- 1975). Few studies have been published suitable, as high doses are required for tively influence fertility and does not gen- regarding the use of oestrogens to prevent several days resulting in pregnancy termi- erally seem to generate long-term uterine pregnancy in bitches. One study showed nation from around day 40. or oestrus abnormalities, more work is

122 Journal of Small Animal Practice Á Vol 50 Á March 2009 Á Ó 2009 British Small Animal Veterinary Association Aglepristone and mid-term pregnancy

required to specifically examine whether stone (RU 534). Journal of Reproduction and Fer- and the effects of a dopamine agonist, cabergo- tility 57 (Suppl), 243-248 line. Journal of Reproduction and Fertility 47, aglepristone treatment influences fertility. GALAC, S., KOOISTRA, H. S., BUTINAR, J., BEVERS,M.M., 403-409 DIELEMAN, S. J., VOORHOUT,G.&OKKENS, A. C. (2000) ONCLIN, K., SILVA,L.D.M.&VERSTEGEN, J. P. (1995) References Termination of midgestation pregnancy in bitches Termination of unwanted pregnancy in dogs with with aglepristone, a P4 receptor antagonist. Ther- the dopamine agonist cabergoline in combination BOWEN, R. A., OLSON, P. N., BEHRENDT, M. D., WHEELER,S. iogenology 53, 941-950 L., HUSTED,P.W.&NETT, T. M. (1985) Efficacy and with a synthetic analog of prostaglandin F2a, GALAC, S., KOOISTRA, H. S., DIELEMAN, S. J., CESTIK,V.& toxicity of estrogens commonly used to terminate either cloprostenol or alphaprostol. Theriogenol- OKKENS, A. C. (2004) Effects of agle´pristone, a pro- canine pregnancy. Journal of American Veterinary ogy 43, 813-822 gesterone receptor antagonist, administered dur- Medical Association 186, 783-788 POST, K., EVANS,L.E.&JO¨CHLE, W. (1988) Effects of ing the early luteal phase in non-pregnant bitches. CONCANNON, P. W., WEINSTEIN, P., WALEY,S.&FRANK,D. Theriogenology 62, 494-500 prolactin suppression with cabergoline on the (1987) Suppression of luteal function in dogs by GOBELLO, C., CASTEX, G., CORRADA, Y., KLIMA, L., DE pregnancy of the bitch. Theriogenology 29, luteinizing hormone antiserum and bromocriptine. LA SOTA,R.L.&RODRIGUEZ, R. (2002) Use of 1233-1243 Journal of Reproduction and Fertility 81, 175-180 prostaglandins and bromocriptine mesylate for SANKAI, T., ENDO, T., KANAYAMA, K., SAKUMA, Y., UMEZU, CONCANNON, P. W., YEAGER, A., FRANK,D.&IYAMILLAI,A. M. & MASAKI, J. (1991) Antiprogesterone com- (1990) Termination of pregnancy and induction of pregnancy termination in bitches. Journal of pound RU 486 administration to terminate preg- premature luteolysis by the antiprogestagen mif- American Veterinary Medical Association 220, epristone in dogs. Journal of Reproduction and 1017-1019 nancy in dogs and cats. Journal of Veterinary Fertility 88, 99-104 HUBLER,M.&ARNOLD, S. (2000) Prevention of preg- Medical Science 53, 1069-1070 FELDMAN, E. C., DAVIDSON, A. P., NELSON, R. W., NYLAND, nancy in bitches with the progesterone antagonist SUTTON, D. J., GEARY,M.R.&BERGMAN, J. G. (1997) T. G. & NUNRO C. (1993) Prostaglandin induction of aglepristone. Schweizer Archiv fur Tierheilkunde Prevention of pregnancy in bitches following abortion in pregnant bitches after misalliance. 142, 381-386 unwanted mating: clinical trial using low dose Journal of American Veterinary Medical Associa- JO¨CHLE, W., LAMOND,D.R.&ANDERSEN, A. C. (1975) oestadiol benzoate. Journal of Reproduction and tion 202, 1855-1858 Mestranol as an abortificant in a bitch. Therioge- Fertility 51 (Suppl), 239-243 nology 4,1-9 FIENI, F., FUHRER, M., TAINTURIER, D., BRUYAS,J.F.& WANKE, M., LOZA,M.E.&MONACHESI,N.&CONCANNON, LINDE-FORSBERG, C., KINDAHL,H.&MADEJ, A. (1992) Ter- DRIDI, S. (1989) Use of PGF2alfa analog, clopros- P. (1997) Clinical use of dexamethasone for termination of mid-term pregnancy in the dog with tenol, for pregnancy termination in dogs. Journal mination of unwanted pregnancy in dogs. Journal of Reproduction and Fertility 39 (Suppl), 332-333 oral RU 486. Journal of Small Animal Practice of Reproduction and Fertility 51 (Suppl), 233-238 FIENI, F., DUMON,C.,TAINTURIER,D.&BRUYAS,J.F. 33, 331-336 WHITEHEAD, M. L. (2008) Risk of pyometra in bitches (1997) Clinical protocol for pregnancy termina- ONCLIN,K.&VERSTEGEN, J. P. (1996) Practical use tion in bitches using prostaglandin F2a. Journal of a combination of a dopamine agonist and a treated for mismating with low doses of oestradiol of Reproduction and Fertility 51 (Suppl), 245-250 synthetic prostaglandin analogue to terminate benzoate. Veterinary Record 162, 746-749 ONE ANKE EBUELTO OZA ESTRE FIENI, F., MARTAL, J., MARNET, P. G., SILIART,B., unwanted pregnancy in dogs. Journal of Small Ani- Z , M., W , M., R , M., L , M., M , BERNARDS, F., RIO, M., BRUYAS,J.F.&TAINTURIER, mal Practice 37, 211-216 J., DUCHENE,A.&CANCANNON, P. (1995) Termination D. (2001) Hormonal variation after early and ONCLIN, K., SILVA, L. D. M., DONNAY,I.&VERSTEGEN,J.P. of pregnancy in dogs by oral administration of mid pregnancy termination in bitches with aglepri- (1993) Luteotrophic action of prolactin in dogs dexamethasone. Theriogenology 43, 487-494

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Theriogenology 70 (2008) 1439–1448 www.theriojournal.com

Induction of abortion with aglepristone signiﬁcantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells H. Kanca a,*, I. Walter b, S. Scha¨fer-Somi c, S. Budik c, S.S. Ay d, I. Kucukaslan a, A.R. Agaoglu a, H. Izgur a, S. Aslan a a Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Ankara, 06110 Ankara, Turkey b Institute for Histology and Embryology, University of Veterinary Science, Veterina¨rplatz 1, 1210 Vienna, Austria c EU Centre for Artiﬁcial Insemination and Embryo Transfer, University of Veterinary Science, Veterina¨rplatz 1, 1210 Vienna, Austria d Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Ondokuz Mayıs, Samsun, Turkey Received 19 December 2007; received in revised form 23 April 2008; accepted 24 April 2008

Abstract In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n = 10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n = 5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n = 7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p < 0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p < 0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC ( p < 0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up- regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/ abortion, which indicates a special role of these cells. # 2008 Published by Elsevier Inc.

Keywords: Aglepristone; Bitch; Endometrium; Estrogen receptor; Pregnancy termination; Progesterone receptor

1. Introduction information about the abortion inducing mechanisms are widely lacking, especially after application of the In the bitch, several medication schemes for the antiprogesterone aglepristone. When compared to induction of abortion have been investigated, however, other medication schemes using dexamethasone [1], prostaglandins [2–6] or combinations of prostaglandins with dopamine agonists [7–9], antigestagens proved to * Corresponding author. Tel.: +90 312 3170315/455; be comparably reliable at all stages of pregnancy and fax: +90 312 3178381. had virtually no side effects [10–16]. Antigestagens E-mail address: [email protected] (H. Kanca). are synthetic steroids that bind with strong afﬁnity to

0093-691X/$ – see front matter # 2008 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2008.04.045 1440 H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 progesterone receptors and competitively displace Retriever, Beagle, German Shepherd, Anatolian Shep- endogenous progesterone [17].Indogs,twoanti- herd, Irish Setter and Bastard), aged 3.6 2.6 years, progestins have been studied, namely mifepristone (RU was included into this study. Most bitches (n = 17) were 468) [11,13] and aglepristone (RU 534), but only introduced to the Clinic of Obstetrics and Gynecology, aglepristone is available for veterinary use and mean- Faculty of Veterinary Medicine, University Ankara (Tr) while has been tested during numerous scientiﬁc because of mismating. In 10 out of 17, abortion was studies. The early administration of aglepristone induced between days 25 and 45 of gestation (group of between days 0 and 25 after mating (n =5)always induced spontaneous resorption/abortion = IRA), the prevented pregnancy. Administration of aglepristone other seven bitches served as controls. The controls between days 26 and 45 after mating (n = 5) induced were healthy pregnant animals and ovariohysterecto- resorption or abortion within 7 days in 96% of cases mized between 25 and 45 on the request of owners. without side effects [15]. During an own study, These animals had no history of abortion or resorbtion. aglepristone was administered at day 30 after mating Additionally, bitches in IRA group were divided into which induced abortion in all of cases (5/5) within 10.6 two subgroups (Group I, n = 5, days 25–35 of days. After this study we concluded that the use of pregnancy; Group II, n = 5, days 36–45 of pregnancy). aglepristone is a safe method for the induction of Gestational age was assessed sonographically accord- abortion, however, the termination of abortion can be ing to Luvoni and Grioni [23]. Five further bitches were delayed when application started after the end of introduced during the same stage of gestation because placentation [18]. of beginning spontaneous resorption and abortion, It has been supposed, that aglepristone induces respectively (group of spontaneous resorption/abor- abortion mainly via its direct effect on the uterus [14], tion = SRA; see Table 1). A detailed history was after competitively displacing P4 and suppressing its taken from all bitches, then clinical and ultrasono- biological effect [19]. Several studies have been graphical examinations performed. After the opera- performed, to investigate the presence and distribution tions, pathosections and histological examinations of estrogen receptors (ER) and PR during physiological of the uteri revealed no speciﬁc infectious or non- canine estrous cycle and pregnancy [20–22]. However, infectious causes. there are no publications available in the relevant literature about the change in the number and distribution 2.2. Induction and observation of resorption/ of PR and ER after application of aglepristone, in abortions the course of resorption and abortion, respectively. Thus, the aim of the present study was to describe and In the IRA groups, abortion was induced by compare the distribution of uterine progesterone and application of 10 mg/kg body weight of the antipro- estrogen receptors in aglepristone-induced and sponta- gesterone aglepristone (Alizine1, Virbac, F) s.c. at 2 neous pregnancy termination in bitches. subsequent days. The start of abortion was diagnosed when bloody vaginal discharge was seen and ultra- 2. Materials and methods sonography revealed characteristic signs of beginning resorption/abortion. Then clinical examinations and 2.1. Animals ultrasonography [24] were performed daily until the end of abortion, when no more fetuses were detected A total of 22 animals from different breeds sonographically (6.0–8.0 MHz; linear, Pie-Medical, (Doberman, Husky, Terrier, Labrador Retriever, Golden Falco 100).

Table 1 Overview over the experimental and control groups Group n Day of gestation Medication Time of OHE Induction of resorbtion/abortion (IRA) Group I 5 25–35 Aglepristone s.c. Immediately after resorption/abortion Group II 5 36–45 Aglepristone s.c. Immediately after resorption/abortion Spontaneous resorbtion/abortion (SRA) 5 25–45 – Immediately after resorption/abortion Control group 7 25–45 – During gestation IRA: induced resorption/abortion; SRA: spontaneous resorption/abortion; OHE: ovariohysterectomy. H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 1441

2.3. Operations and sampling microscope at 400 magnification according to Vermeirsch et al. [21,22]. For the assessment of the Animals were ovariohysterectomized under general total and proportional scores (PR and ER), 100 positive anesthesia. Immediately after extirpation of the organs, cells per slide were evaluated and grouped into an tissue samples were cut off from different sites of the intensity and proportional score. For the PR, the method uterus: placental sites, interplacental sites and corpus after Vermeirsch et al. [22] was used (intensity score: uteri. One half of the samples was covered with Tissue 0 = no staining, 1 = weak, 2 = moderate, 3 = strong, 1 Tek , then stored in liquid nitrogen at 196 8C for later 4 = extremely strong staining; proportional score: RNA isolation. The other half was fixed in a 4% formalin 0 = no positive nuclei, 1 = <1%, 2 = 1–9%, 3 = 10– solution, then embedded in paraffin and histosections 32%, 4 = 33–65%, 5 = >65% positive nuclei). For the prepared. In all groups, before the operation, blood ER, the method according to Vermeirsch et al. [21] was samples were taken once, serum produced and stored at used (intensity score: 0 = no staining, 1 = weak, 18 8C until analyses for estradiol and progesterone 2 = moderate, 3 = strong staining; proportional score: concentrations. equal). The intensity score indicates the subjective intensity of the staining, the proportional score the 2.4. Immunohistochemistry (IHC) percentage of positively stained cells. Both scores were taken together and a total score calculated. Prior to the For the immunohistochemical examinations, the cell count, the thickness of the endometrium and following antibodies were used: Anti-ER (1:200 Rabbit myometrium was measured in four different micro- monoclonal, Zymed Laboratories, South San Fransisco, scopic fields in each histological section using the USA); Anti-PR (1:50 Rabbit monoclonal SP2, NeoMar- computer image analyzing program (Eclipse; Nikon, kers, Fremont, CA, USA) and staining prepared as Austria). The samples were obtained from placental and follows: serial paraffin sections were cut at 3 mm interplacental sites of the uterus and the corpus uteri. thickness and placed onto APES (3% 3-aminopropyl- triethoxysilane, Sigma A3648, dissolved in 100% 2.5. Polymerase chain reaction (RT-PCR) acetone) coated slides. After deparaffinizing in xylene and rehydration, endogenous peroxidase activity was For qualitative RT-PCR, pieces of ovaries and uteri inhibited by 0.6% methanol/perhydrol for 15 min. (placental sites, interplacental sites and corpus uteri) Microwave pretreatment (3–5 min in 0.1 M EDTA were homogenized using TRI Reagent (T 9424, Sigma– solution, pH 8.0) was performed in the aim of antigen Aldrich, A) and subsequently treated as recommended retrieval. All following incubation steps were performed by the manufacturer. With TRI Reagent, it is possible to in an immunostaining center (Sequenza; Shandon, isolate mRNA, protein and DNA of any parameter Pittsburgh, PA, USA). After rinsing with PBS, the chosen. sections were incubated with normal goat serum (1.5%) For the detection of PR-genes, primers were designed for 30 min at room temperature, to prevent nonspecific using the sequences available from the Nucleotide binding reactions. Subsequently, primary antibodies database (PR: sense 50 CAG GTG TAC CAG CCG TAC were used by incubating overnight at 4 8C. A secondary CT 30, antisense 50 ATT TCG AAA ACC TGG CAATG antibody with a horseradish peroxidase (HRP)-conju- 30, 618 bp, accession no. AF177470). The products were gated dextran polymer to enhance the signal was used visualized after separation by ethidium bromide contain- (PowerVision Poly-HRP anti-rabbit IgG; Immunovision ing agarose gels using UV-light. The bands of the Technologies, Daly City, CA, USA). Finally, sections expected size were cut out of the agarose gel and sent to were washed with PBS and peroxidase activity detected the sequencing service (IBL, Vienna, A) using one of the with DAB substratum (10 mg 3,30-diaminobenzidine in primers used for amplification for the sequencing 50 ml 0.1 M Tris buffer, pH 7.4) and 0.03% (v/v) H2O2 procedure. The sequence obtained was compared with for 10 min at room temperature. After that, sections were the databases using BLAST analysis. The mastermix counterstained with hemalumn, dehydrated and mounted served as negative control. with DPX (Fluka, Buchs, Switzerland). Negative controls included the replacement of the primary 2.6. RNA quality control antibody by PBS and an IgG control by using unspecific rabbit serum instead of the primary antibody. In each sample, the quality of the isolated RNA was The expression of progesterone (PR)- and estrogen assessed via measurement of the RNA integrity number receptors (ER) in uterine tissue was evaluated in a light (RIN). The RIN values were measured with the Agilent 1442 H. Kanca et al. / Theriogenology 70 (2008) 1439–1448

2100 bioanalyzer on microfabricated RNA Lab Chip 3. Results kits (Agilent technologies GmbH, Vienna, A). Among others, the bioanalyzer software generates an electro- 3.1. Histological findings pherogram which provides a detailed picture of the size distribution of the RNA fragments and allows thus a After spontaneous and induced abortions, in the visual assessment of the quality of an RNA sample. glandular chambers of the endometrium and the placenta, accumulations of secretions within the glandular cham- 2.7. Statistical analysis bers and degenerative alterations of the tissue were detected (Fig. 1). Data are given as means standard deviations Measurement of the uterine tissue revealed, that (X S.D.). All statistical comparisons were performed the endometrium in the IRA group was 1260.9 using the SPSS software1 (Version 11 for Windows, 165.8 mm, which was significantly thicker than in the SPSS Inc., Chicago, IL, USA). Comparisons of two control group (873.5 114.8 mm; p < 0.05). In the mean values within one group were performed using the SRA group, the endometrium was also thicker than in Wilcoxon test (respectively, Fisher exact test) and the controls, however, the difference was non significant comparisons between groups with the Mann–Whitney (SRA: 1129.6 515.7 mm). U-test. Comparisons of more than two independent groups were performed using the Kruskal–Wallis Test. 3.2. Progesterone receptor A p-value of <0.05 was considered statistically significant. Since the total score contains the subjec- Comparison of total scores with the number of tively assessed intensity score, each calculation was positively stained cells revealed no statistical significant repeated using the number of positively stained cells differences. Therefore, in the present study, results are solely and the results compared. expressed using the total score. In the placental part of the uterine horns, brown nuclear staining demonstrating the presence of PR was detected in myometrial smooth muscle cells (MSMC), endometrial stroma cells (ESC), endometrial glandular epithelia (EGE) and glandular chamber epithelial cells (GCE). However, in the blood vessels, no PR were detected (Fig. 2A–C). In all groups (IRA, SRA and controls), the average total scores of the progesterone receptors (TPR) in the ESC and the MSMC were significantly lower than thoseintheEGEandtheGCE(Table 2; p < 0.05). In addition, the TPR in ESC was significantly lower in the control group than in the SRA- and IRA- group, respectively (5.8 vs. 6.5 and 6.7; p < 0.01; Fig. 3). At the interplacental sites of the uterine horn and the corpus uteri, the distribution of PR resembled the placentation sites. In the control group, the average TPR was significantly lower in ESC (5.6) than in all other cell types (SRA: 6.6; IRA: 6.6; p < 0.05), with the lowest value in the corpus uteri (control: 5.3, IRA: 6.3, SRA: 6.6; p < 0.01). In the control and IRA group, the average TPR were highest in epithelial cells (EGE, ESE, GCE; see Table 2). Groups I and II (IRA), in which pregnancy was terminated on different days, did not differ significantly Fig. 1. H & E staining of a placental specimen after induction of concerning any cell population in any part of the uterus, abortion. Note the high amount of secretions degenerated cells in the except the MSMC in the interplacental part of the uterus glandular chambers (GC). Scale bar = 50 mm. (Group I: 7.0; Group II: 6.5). H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 1443

Fig. 2. Immunohistochemical demonstration of PR in the normal placenta (A) and after induced (B) and spontaneous (C) abortion. MM: myometrium; UG: uterine glands. Scale bar = 50 mm.

Table 2 Total scores of the progesterone receptor (TPR) in (a) placental and (b) interplacental part of the uterine horn—all groups Group MSMC EGE GCE p (a) SRA (n = 5) 6.7 0.2a (6.2–6.9) 7.2 0.1b (7.0–7.4) 7.0 0.1b (6.9–7.5) <0.05 IRA (n = 10) 6.8 0.4a (6.0–7.6) 7.2 0.2a,c (6.9–7.8) 7.1 0.2c (6.8–7.5) <0.05 Controls (n = 7) 6.8 0.3a (6.1–7.1) 7.1 0.1a,c (6.9–7.2) 7.1 0.0c (7.0–7.2) <0.05 (b) SRA (n = 5) 6.6 0.3a (5.9–6.8) 7.1 0.1b (6.8–7.4) 7.0 0.1b,c (6.9–7.2) <0.05 IRA (n = 10) 6.7 0.4a (6.0–7.1) 7.1 0.2b (6.7–7.4) 7.1 0.0b (7.0–7.6) <0.05 Controls (n = 7) 6.8 0.3a (6.1–7.0) 7.1 0.1a (6.9–7.2) 7.1 0.1a (6.8–7.2) <0.05 MSMC: myometrial smooth muscle cells; EGE: endometrial glandular epithelia; GCE: glandular chamber epithelial cells; ESE: endometrial surface epithelia; SRA: spontaneous resorption/abortion; IRA: induced resorption/abortion; controls: normal pregnant bitches. Values with different indices (a–c) within one line differ signiﬁcantly ( p < 0.05).

3.3. Estrogen receptors group, the TER was significantly lower than in MSMC and ESC of the controls (6.2 and 6.0, p < 0.05, Fig. 5), Like the PR, the estrogen receptors (ER) were whereas no difference between groups was detectable detectable in the placental (Fig. 4A–C) and inter- concerning the epithelial cells. placental part of the uterine horn and in the glandular chambers. Positive immune reactions for ER were detected in all cell types investigated (MSMC, ESC, EGE, GCE). In the placental part of the uterine horn and in all groups, the average score for TER was significantly lower in MSMC and ESC than in EGE and GCE ( p < 0.05 and p < 0.01, see Table 3). Comparison between groups revealed no significant differences concerning any cell population ( p > 0.05). Similarly, at the interplacental part, the TER was significantly lower in MSMC and ESC (5.7 and 3.8) Fig. 3. Total scores of the progesterone receptor (TPR) in ESC of the placental and interplacental part of the uterine horn. IRA: induced than in EGE- and ESE-cells (6.3 and 6.4; p < 0.05 and resorption/abortion; SRA: spontaneous resorption/abortion; control: p < 0.01), in all groups investigated (Table 3). How- normal pregnant animals. **Bars with indices differ significantly ever, in MSMC (5.7) and ESC (3.8 and 5.0) of the SRA- ( p < 0.01). 1444 H. Kanca et al. / Theriogenology 70 (2008) 1439–1448

Fig. 4. Immunohistochemical demonstration of ER in the normal placenta (A) and after induced (B) and spontaneous (C) abortion. MM: myometrium; UG: uterine glands. Scale bar = 50 mm.

In the corpus uteri, like in the interplacental part of the group of spontaneous abortions (SRA), in one bitch the uterine horn, the TER for MSMC and ESC (4.9 and there was still PR-mRNA detectable. In the control 4.1, SRA; 5.0 and 4.3, IRA; p < 0.01) were signiﬁcantly group, mRNA for PR was assessed in all bitches and at lower than for EGE- and ESE (5.9 and 5.4, SRA, all sites investigated. The RIN value of the mixed p < 0.05; 6.1 and 6.1, IRA, p < 0.01). However, in the sample from the control group was 10. control group, signiﬁcantly higher TER for MSMC and ESC were detectable than in all other groups (MSMC: 3.5. Serum concentrations of progesterone and 5.6, ESC: 5.2; p < 0.05). estradiol-17ß

3.4. RT-PCR (progesterone receptors) In normal pregnant bitches, who had been ovariohysterectomized between days 25 and 45 of gestation, Quality assessment of the samples of the IRA and the average P4 value was 78.8 nmol/l, which was SRA group revealed very low RIN values, with an signiﬁcantly higher compared to SRA and IRA (13.5 average of 2.25 0.72 (0–2.6). In the group of induced and 24.3 nmol/l, respectively, p < 0.01 each). In the abortions (IRA), no mRNA for PR was found by means controls, the average serum E2 concentration was of RT-PCR, neither in the placental and interplacental 18.6 pg/ml; in the abortion groups, the respective values part of the uterine horn, nor in the corpus uteri. Only in were 24.8 (SRA) and 33.4 pg/ml (IRA, p > 0.05).

Table 3 Total scores of the estrogen receptor (TER) in (a) placental and (b) interplacental part of the uterine horn Group MSMC ESC EGE GCE p (a) SRA (n = 5) 5.7 0.7a* (5.1–6.5) 4.8 0.6a,b* (4.0–5.3) 6.5 0.0a,c* (6.5–6.5) 6.6 0.4c* (5.7–6.8) <0.05 IRA (n = 10) 5.8 0.6a* (4.5–6.6) 5.0 0.5b* (4.1–5.6) 6.5 0.2c* (6.0–7.0) 6.6 0.4c* (5.8–6.9) <0.01 Controls (n = 7) 5.9 0.6a* (5.0–6.6) 5.3 0.5a,b* (4.5–6.2) 6.4 0.1a,c* (6.3–6.6) 6.7 0.1c* (6.5–6.9) <0.05 p >0.05 >0.05 >0.05 >0.05 (b) SRA (n = 5) 5.7 0.7a* (5.1–6.5) 3.8 1.1a,b* (2.4–5.1) 6.3 0.4a,c* (5.6–6.6) 6.4 0.5c* (5.8–6.8) <0.05 IRA (n = 10) 5.7 0.4a* (5.1–6.3) 5.0 0.3b** (4.4–5.4) 6.5 0.1c* (6.4–6.8) 6.6 0.3c* (5.8–6.9) <0.01 Controls (n = 7) 6.2 0.3a** (5.6–6.5) 6.0 0.4a,b** (5.6–6.5) 6.2 0.1a,c* (6.0–6.4) 6.5 0.3c* (5.8–6.7) <0.05 p <0.05 <0.05 >0.05 >0.05 MSMC: myometrial smooth muscle cells; ESC: endometrial stroma cells; EGE: endometrial glandular epithelia; GCE: glandular chamber epithelial cells; ESE: endometrial surface epithelia; SRA: spontaneous resorption/abortion; IRA: induced resorption/abortion; controls: normal pregnant bitches. Values with different indices (a–c) within one line differ significantly and highly significantly, respectively ( p < 0.05 and p < 0.01). *,**Values with different indices within one row differ significantly ( p < 0.05). H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 1445

intensity and found no significant difference between the two methods. Our results, presented as scores, are thus reliable. In accordance to Galabova-Kovacs et al. [30] and Vermeirsch et al. [22] we detected PR and ER in the luminal and glandular epithelium, in stromal cells, and in the myometrial smooth muscle cells, as reported in other species [31–33]. Biochemical [34,35] and immunohistochemical [21,22,36] studies in the dog revealed cyclic changes in the PR and ER expression Fig. 5. Total scores of the estrogen receptor (TER) in ESC of the placental and interplacental part of the uterine horn. IRA: induced throughout the estrous cycle. Generally, during the resorption/abortion; SRA: spontaneous resorption/abortion; control: estrus cycle, stromal cells were found to have higher normal pregnant animals. **Bars with indices differ significantly staining scores for PR than epithelial cells, whereas ( p < 0.01). stromal cells were found to show more fluctuations in the ER expression [21,22]. Regulation of cyclic 4. Discussion endometrial changes is believed to be mediated by steroid hormones, namely the estradiol-17ß: progester- 4.1. Embryonic/fetal death one ratio, and their effects on stromal cells. During the cycle, staining scores for PR and ER in the uterine horns Maternal endocrine abnormalities, immune factors, were found to be high during proestrus, and expression genetic factors, environmental factors, and nutritional of both receptors decreased through estrus to early factors have to be considered as causes for embryonic/ metestrus. During late metestrus and anestrus, while fetal death in dogs [25]. Whether the fetus is resorbed or serum P4 concentrations already decreased or were aborted depends on the cause of pregnancy loss, stage basal, staining scores for ER and PR in the stromal and of gestation, and maternal and fetal responses [26] smooth muscle cells increased again [21,22].In Spontaneous, non-infectious resorptions can be caused humans, ER and PR expression in the endometrium by large sized puppies [26] or uneven distributed puppies changed similarly during the menstrual cycle [42,43]. in the uterine horns [24]. In addition, inhomogeneous The relative intense expression of PR in stroma in the distribution of estrogen receptors (ER) in the uterine late secretory phase indicates its role for the support of tissue has been described as a possible cause. In these decidualization during early pregnancy [43]. cases, nidation and placentation can take place; however, In another study, in normal pregnant bitches, as during the number of receptors might not be sufficient for the the cycle, stromal cells stained more frequently positive maintenance of pregnancy [27,28]. In the present study, for ER-alpha and PR than epithelial cells [44]. However, the bitches were introduced to the clinic immediately in the present study, in all groups at placental as well as after abortion, information about size and distribution of interplacental sites, the PR-score was significantly lower puppies is therefore lacking. After both induced and in MSMC and ESC than in the EGE and GCE ( p < 0.05). spontaneous resorption/abortions we found accumula- The difference could be due to different phases of tions of secretions and degenerative alterations in the gestation at the time of sampling since this was not glandular chambers of the endometrium and the placenta; indicated by the cited authors. these morphological changes might have contributed to Concerning the comparison between groups, we embryonic/fetal death [29]. found significantly higher TPR scores in ESC after resorption/abortion than in normal pregnant animals, 4.2. Regulation of receptor expression in indicating an up-regulation of PR during resorption/ endometrium and placenta abortion. To our knowledge, this is the first report on the effects of progesterone antagonists on the abundance In the present study, the total progesterone receptors and the distribution of uterine steroid hormone receptors (TPR) and total estrogen receptors (TER) were assessed in canine species. Similarly, in pregnant women, the according to the method of Vermeirsch et al. [21,22]. expression of PR and ER in endometrium and decidua These authors used a score, calculated of cell number and increased in the endometrial stroma and glandular subjectively assessed staining intensity. Since results epithelium after mifepristone application [37,38].In might differ considerably between investigators, we contrary, application of different agents with anti- additionally evaluated the cell number without staining progesterone effect decreased PR and ER in the uterus 1446 H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 from early pregnant mice [39]. This points to species- in the abortion groups IRA and SRA ( p < 0.01 each); specific receptor regulation during induced resorption/ the E2 concentrations did not differ between groups abortion. Whether this is dependent on phase of ( p > 0.05). Vermeirsch et al. [21] detected a negative gestation, uterine site and/or progesterone and estradiol correlation between the serum P4 concentration and the concentration, is not proven. expression of ER in the corpus uteri in pregnant bitches. The present results revealed that the TER score in ESC In addition, a positive effect of E2 and a negative at interplacental sites was significantly lower in the SRA influence of P4 on the respective receptors has been group than in the IRA and control group ( p < 0.05), this reported in the dog and other species [20,31]. The difference was non-significant at placental sites, how- down regulating effect on the PR is absent when P4 ever, the loss of ER might have contributed to embryo/ concentrations in serum are low or serum concentra- fetal loss [27,28]. In the normal pregnant controls, the tions of E2 are increasing [35]. The markedly lower TER values in MSMC and in ESC were significantly serum P4 concentration observed in abortion groups higher than in the abortion groups. This was also the case compared to the normal pregnant controls is a well in ESC of the corpus uteri ( p < 0.05), indicating a described phenomenon after application of PR antago- general down-regulation during resorption/abortion, nists [11,14,18] supposed to be caused by fetal death. however, this was more pronounced in the spontaneous This hypothesis is confirmed in the present study, since cases. In contrast to these results, other authors found an the significant decrease in P4 and fetal death occurred at increase in the number of ER in the endometrium of the same time. However, it is well known that after pregnant women and rhesus monkeys after application of application of P4 antagonists, serum P4 concentrations mifepristone [40,41]. This confirms our hypothesis, that are not decreased within 24–48 h after its administra- in the bitch, ER and PR expression during spontaneous tion, due to delayed luteolysis [15,18]. In addition, the and induced resorption/abortion is regulated in a species- serum E2 concentrations did not change significantly specific way and independent of the time of induction after application of aglepristone but corresponded to since no differences were assessed between Group I normal pregnancy as described before [18]. (induction between days 25 and 35) and Group II (induction between days 36 and 45). It is known that 5. Conclusions aglepristone induces abortion by blocking progesterone receptors inside the uterus [17,45], however, the local In the present study, we found that induction of effect on the steroid receptors has not been described yet. abortion had a significant effect on the expression of PR The results of the present study indicate that aglepriston and ER in endometrial stromal cells. Consequently, even enhances the expression of stromal PR in the uterus and though higher numbers of receptors were found in the maternal placenta, which might be due to the decrease in the biological effect of P4, since results were similar in epithelial cells, endometrial stromal cells may be more important in the progress of resorption/abortion after the IRA and SRA group. induction with aglepristone. However, the factors regu- In the present study, detection of PR mRNA in lating uterine steroid hormone receptor expression during tissues obtained from animals after resorption/abortion normal pregnancy and resorption/abortion, respectively, was not possible. According to the sample quality have to be further investigated in the bitch. assessment via measurement of the RNA integrity number (RIN), the mRNA content and quality of the samples of the IRA and SRA groups was inferior. As the References mRNA content and quality of the control group was good, we can exclude a methodological error during [1] Austad R, Lunde A, Sjaastad OV. Peripheral plasma levels of sampling and subsequent RNA preparation. We oestradiol-17-beta and progesterone in the bitch during oestrous suppose, that due to advanced stages of resorption/ cycle, in normal pregnancy and after dexamethasone treatment. J Reprod Fertil 1976;46:129–36. abortion the mRNA in the uterine tissue was too [2] Concannon PW, Hansel W. Prostaglandin F2alpha induced degenerated for the here used RT-PCR method. luteolysis, hypothermia, and abortions in beagle bitches. Pros- taglandins 1977;13:533–42. 4.3. Serum concentrations of progesterone and [3] Vickery B, McRae G. Effect of a synthetic prostaglandin analo- estradiol-17ß gue on pregnancy in beagle bitches. Biol Reprod 1980;22: 438–42. [4] Romagnoli SE, Cela M, Camillo F. Use of prostaglandin F2 In the present study, the average serum P4 con- alpha for early pregnancy termination in the mismated bitch. Vet centrations were significantly higher in the controls than Clin North Am Small Anim Pract 1991;21:487–99. H. Kanca et al. / Theriogenology 70 (2008) 1439–1448 1447

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Repeated induction of abortion in bitches and the effect on plasma concentrations of relaxin, progesterone and estradiol-17b S. Scha¨fer-Somi a,*, O.A. Aksoy b, H.B. Beceriklisoy b, A. Einspanier c, H.O. Hoppen d, S. Aslan b a Clinic for Obstetrics, Gynecology and Andrology, University of Veterinary Medicine, Veterina¨rplatz 1, 1210 Vienna, Austria b Clinic for Obstetrics and Gynecology, Faculty of Veterinary Medicine, University Ankara, 06110 Diskapi, Turkey c Veterinary-Physiological-Chemistry Institute, University Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany d Section for Chemical Analytics and Endocrinology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, Haus 123, 30173 Hannover, Germany Received 6 February 2007; received in revised form 14 June 2007; accepted 14 July 2007

Abstract The aim of the present study was to investigate the effects of two medications on two subsequent abortions and plasma hormone concentrations of dogs. For this purpose, two groups of bitches (n = 5 each), received the antiprogesterone aglepristone (Alizine1) at 10 mg/kg body weight on two subsequent days around day 30 after mating. In group II, the antiprolactin cabergoline (Galastop1) was additionally administered po at 5 mg/kg body weight until the start of abortion. The plasma concentrations of relaxin, progesterone (P4) and estradiol-17b (E2) were measured before, during and after each abortion. During the next cycle after the abortion, the same bitches were mated again and in pregnant animals, induction of abortion was performed as before. During the third cycle, pregnant bitches were allowed to whelp. Termination of first pregnancy occurred significantly earlier after the combined treatment (6.8 versus 10.6 days, p < 0.05). In both groups and during both abortions, relaxin varied between individuals; however, there was a continuous decrease after the abortions and no significant differences between groups ( p > 0.05). In one bitch with high relaxin concentrations before treatment (11.6 ng/ml), a cystic endometrial hyperplasia was diagnosed. In the aglepristone only group, P4 concentrations increased significantly after the first application ( p < 0.05), then decreased continuously until day 45 after the beginning of abortion. In the combined group, there was a continuous decrease until day 45 ( p > 0.05). At this time, P4 concentrations between 0.47 and 84.9 nmol/l were measured in both groups. The level of E2 over time was not influenced by any medication. We therefore note that the two medications mainly influenced plasma concentrations of P4 in different ways, probably due to specific treatment-hormone interactions. However, all measurements fell within the range considered normal. # 2007 Elsevier Inc. All rights reserved.

Keywords: Dogs; Pregnancy termination; Fertility; Hormones

1. Introduction

* Corresponding author at: Department for Animal Breeding and Termination of pregnancies in bitches is often Reproduction, Clinic for Obstetrics, Gynaecology and Andrology, required, especially in cases of mismating and University of Veterinary Medicine Vienna, A-1210 Vienna, Austria. Tel.: +43 1 25077 5412; fax: +43 1 25077 5490. miscarriage. A single mismating and induced abortion E-mail address: [email protected] usually has no harmful consequences, provided the (S. Scha¨fer-Somi). abortion is professionally carried out and carefully

0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.07.012 890 S. Scha¨fer-Somi et al. / Theriogenology 68 (2007) 889–895 observed [1,2]. However, no data are available namely, with aglepristone and a combination of concerning fertility after two or more successive aglepristone and cabergoline. In addition, we monitored abortions and whether or not fertility relates to the the effects of this combination on the course of plasma medication scheme and/or endocrine changes during concentrations of P4, E2 and relaxin, and observed the and after the abortions. course of consecutive cycles and abortions. Several methods exist for the induction of abortions in dogs. As one of the first trials, dexamethasone was 2. Animals and methods administered orally twice daily for 10 days from day 30 of pregnancy on. This resulted in abortion in the two 2.1. Animals treated bitches [3]. However, side effects after the use of dexamethasone were polydipsia, polyuria, vaginal Ten dogs of different breeds (two German shepherds, discharge, restlessness, anorexia and emesis [4]. Later two Anatolian shepherds, five malinois, one dalmatian), natural and synthetic prostaglandins were used during aged 2–6 years, with an average body weight of all stages of pregnancy, at different dosages and with 20.7 Æ 4 kg (17–28 kg), were randomly allotted to two different application regimens [5–9]. To further experimental groups. The dogs were housed in the improve the effect and to enhance abortion, natural kennel of a military working dog breeding unit in and synthetic prostaglandins have been combined with Gemlik (Tr). Each bitch was housed in a box (3 m2) the dopamine agonist cabergoline [10–12] or with with a paddock (9 m2). They all received commercially bromocriptine mesylate [13]. Cabergoline suppresses available canned food and water ad libidum. pituitary prolactin secretion and is supposed to antagonize the luteotrophic effect of prolactin. How- 2.2. Treatments, blood sampling and observation of ever, strong side effects like vomiting, diarrhea, and abortions decrease in blood pressure have been described after the use of prostaglandins. These side effects were less Group I (n = 5): estrus was detected by means of pronounced with synthetic prostaglandins [10].How- clinical symptoms, vaginal cytology and P4 measure- ever, antigestagens proved to be most reliable at all ments (enzyme immunoassay, EIA). A progesterone stages of pregnancy and had virtually no side effects value between 15 and 30 nmol/l indicated the day of [14–21]. Antigestagens are synthetic steroids that bind ovulations. All dogs were mated one or 2 days after with strong affinity to progesterone receptors and ovulations and pregnancy was confirmed by ultrasono- competitively displace endogenous progesterone [22]. graphic examination around day 30 after mating. In dogs, two anti-progestins have been studied, namely Immediately afterwards, the dogs were treated sub- mifepristone (RU 486) [15,17] and aglepristone (RU cutaneously with aglepristone (Alizine1) at a dose of 534), but only aglepristone is available for veterinary 10 mg/kg body weight on two consecutive days. Blood use. In one study, the early administration of samples were collected before aglepristone adminis- aglepristone between days 0 and 25 after mating tration and every second day until the termination of always prevented pregnancy. Administration of agle- abortion. The start of abortion was diagnosed when pristone between days 26 and 45 after mating induced bloody vaginal discharge was seen and ultrasonography resorption or abortion within seven days in 96% of cases revealed characteristic signs of beginning resorption/ without side effects [20]. This suggested that the use of abortion. Then clinical examinations and ultrasono- aglepristone is a safe method for prevention of graphy were performed daily until the end of abortion, pregnancy and induction of abortion before day 45 of when no more fetuses were detected sonographically. gestation. During the second half of pregnancy, Further blood samples were collected at days 1, 2, 15, antigestagens can be combined with prostaglandins, 30 and 45 after the abortions. The same dogs were and this has proved to be effective for inducing mated during the following cycle and another abortion parturition in dogs [23,24]. However, a combination of a induced with the same treatment and procedure as local receptor blocker (aglepristone) with an indirect before. During the subsequent third cycle, dogs were luteolytic drug (cabergoline) might terminate preg- mated again and pregnant animals were allowed to nancy in a comparable time or even quicker while whelp. avoiding the side effects usually seen when prostaglan- Group II (n = 5): Bitches were examined, mated and dins are used. pregnancy confirmed as described for group I. Thus, in the present study, we induced abortions Immediately after confirmation of pregnancy, dogs repeatedly with two different treatment schemes, were treated with a combination of 10 mg/kg body S. Scha¨fer-Somi et al. / Theriogenology 68 (2007) 889–895 891 weight aglepristone (Alizine1) subcutaneously and 3.2. Relaxin 5 mg/kg body weight cabergoline (Galastop1) orally on two consecutive days. The treatment was continued In group I, relaxin concentrations ranged between with cabergoline alone until the beginning of abortion. 0.21 and 1.33 ng/ml before treatment. Only one bitch Blood sampling and the observation of abortion was had an exceptional high value (11.6 ng/ml); in this performed as in group I. The whole procedure was bitch, a cystic endometrial hyperplasia was diagnosed repeated during the following cycle. During the third later. In group II, the average relaxin concentrations cycle, pregnant bitches were allowed to whelp. tended to be higher (0.59–13.25 ng/ml before treatment), however, at no time did they differ signifi- 2.3. Hormone measurements cantly between groups ( p > 0.05). The course of relaxin was similar in both groups and during both For the production of plasma, blood samples were abortions and showed a more or less continuous centrifuged three minutes at 3000 Â g. Samples were decrease after the abortion; at day 45 after the stored at À18 8C until analysis. The concentrations of termination of abortion, relaxin concentrations ranged P4 and E2 in peripheral blood plasma were measured between 0.15 and 10.8 ng/ml. In both groups, the using radioimmunoassay [25]. The concentrations of average relaxin concentrations did not differ sig- relaxin in peripheral blood plasma were measured using nificantly between first and second abortion a modified species-specific EIA system [26]. ( p > 0.05; Fig. 1a and b).

2.4. Statistical analyses 3.3. Progesterone and estradiol-17b

Data are given as means Æ standard deviations The average P4 and E2 concentrations did not differ (x Æ S.D.). All statistical comparisons were performed significantly between groups ( p > 0.05; Figs. 2 and 3). using the SPSS software1 (Version 14 for Windows, However, in group I, the average E2 and P4 SPSS Inc., Chicago, IL, USA). Comparisons of two concentrations tended to be higher before and during mean values within one group were performed using the the first abortion than during the second abortion Wilcoxon test (resp. Fisher exact test) and comparisons (Figs. 2a and 3a). During both abortions, in group I, the between groups with the Mann–Whitney–U-test. A p- average plasma concentrations of P4 increased sig- value of <0.05 was considered statistically significant. nificantly at 48 h after aglepristone administration (from 99.5 to 162.8 nmol/l, first abortion, p < 0.05), 3. Results and decreased in group II (from 74.4 to 64.5 nmol/l, first abortion, p > 0.05; Fig. 3a and b). 3.1. Clinical data 4. Discussion Clinical data are displayed in Table 1. In both groups, the time between start of medication and termination of In the present study, we induced abortions with two abortion, as well as the interestrous interval, did not different medications repeatedly. Starting at mid differ significantly between the first and second abortion gestation, one group of bitches was treated with the ( p > 0.05). However, after induction with alizine and progesterone antagonist aglepristone, another with a cabergoline, the first abortion terminated significantly combination of aglepristone and the antiprolactin quicker than after induction with alizine alone (6.8 cabergoline. As far as the course of abortion is versus 10.6 days, p < 0.05). concerned, there were no significant differences We note the following additional information. In between groups, except a significant earlier start of group I, after two abortions, one bitch was mated during abortions with the combined treatment after first the following three cycles and never became pregnant medication. During an earlier study [18], dogs were (interestrous intervals: 93, 112 and 195 days). This mated 1–2 days after ovulations, medication with bitch was ovariohysterectomized and a cystic endo- aglepristone alone started 30 days after ovulations and metrial hyperplasia diagnosed pathologically and termination of abortions occurred 4–7 days later, which histologically. In group II, during the second abortion, is comparable to our results with the combined one bitch aborted all puppies except one, that had to be treatment. However, in the present study, medication delivered by caesarean section; this bitch was not mated with aglepristone alone started between days 25 and 37 again. after mating and in some cases it took more time than 892 S. Scha¨fer-Somi et al. / Theriogenology 68 (2007) 889–895

the expected 3–7 days, when abortion was induced after completion of placentation. % bitches pregnant Fertility after subsequent abortions has not been = 2) 50 (1/2)

= 3) 80 (3/4) investigated yet. However, it was not inﬂuenced by up to n n two abortions in the aglepristone group. Unfortunately, in the group with combined treatment, only two bitches came into heat after the second abortion, which is not S.D.

Æ enough for a reliable conclusion concerning fertility. 77.7 (86–260) ( 33.2 (103–150) ( x Æ Æ However, after the ﬁrst abortion results are comparable in both groups. All whelpings were completely normal Interestrous interval days with average to high puppy numbers. During a recent

= 3) 126.5 study [27], induction of abortion with a long-acting n prostaglandin F(2a) analogue, fenprostalene, starting = 3) 153

n on day 25 after ovulation, led to low conception rates S.D.

Æ during the subsequent estrus (50%). In our study, 0.0 ( 1.2 (8–10) ( x Æ Æ conception rates were higher in both groups.

Termination of abortion 2 after days During the present study, relaxin concentrations varied considerably between individuals, however, they

S.D. agreed with those reported in the literature [28,29]. Æ x 2.6 (32–37) 8.0 1.7 (30–34) 8.7 According to other researchers, relaxin concentrations Æ Æ are not related to the number of embryos inside the Start of medication 2 at day of gestation uterus [28,30]; however, concentrations are known to increase between days 28 and 33 of gestation due to % bitches pregnant = 5) 80 (4/5) 35 = 5) 60 (3/5) 31.7 n n S.D. Æ 24.2 (74–138) ( 58.5 (79–233) ( x Æ Æ 0.05). < p Interestrous interval days 1.5 a (9–12) 115 1.3 b (6–8) 133.6 Æ Æ S.D. days Æ Termination of abortion 1 after x = 5) 10.6 = 5) 6.8 n n S.D. of Æ 3.0 (25–33) ( 2.2 (29–35) ( x Æ Æ gestation Start of medication 1 at day S.D. 4.7 (17–28) 28.4 3.0 (17–25) 31.6 Æ Æ Æ x Fig. 1. Average relaxin values after induction of abortion with Alizine kg (a) and Alizine + cabergoline (b), before and during abortions. Abor- t. = Abortion. 1, 2 Alizine = 1st and 2nd application of Alizine Values Table 1 Clinical data from both groups, beforeGroup and during abortions Weight 1 and 2 Different letters in the same column indicate statistically significant differences ( 1 21.6 2 20.4 in Fig. 2a were calculated without the bitch Aslik. S. Scha¨fer-Somi et al. / Theriogenology 68 (2007) 889–895 893 ripening of the placenta. In the present study, some cardiovascular cases [32,33], might explain this abortions were induced before this increase, which phenomenon. The H2 gene relaxin contains a gluco- might have caused the high individual variations at the cortoid-responsive element [34], therefore, relaxin beginning of medications. expression seems to be influenced by glucocorticoids. Only one bitch had outstandingly high values of As an abortifacient, aglepristone acts like a relaxin before and throughout her first abortion, progesterone antagonist inside the uterus and does probably resulting from her endometrial cystic hyper- not have direct and immediate luteolytic properties. plasia. In all other bitches, no clinical problems Aglepristone does not modify plasma concentrations of occurred during the period of the study, even though P4, prostaglandins, oxytocin or cortisol within 24 h comparably high concentrations were measured in after administration. In the present study, P4 concen- individual bitches. We therefore assume, that the relaxin trations increased 48 h after first medication in the concentrations measured here were within the bound of Alizine group, which is comparable to the results of normal, however, in some cases high values might Fieni et al. [35,36]. Values decreased thereafter in indicate a uterine pathology. Interestingly, in rats with a accordance with the results of Galac et al. [18] and Fieni pathological uterus during pseudopregnancy, relaxin et al. [20]. After abortion, P4 concentration usually levels were also elevated more than 20-fold compared to declines within 8–34 days to basal concentrations. normal cycling rats [31]. Relaxin increases intracellular However, plasma P4 concentrations have been observed cAMP in the endometrium of different species (human, to remain high for some time after the abortion monkey, rat), which is an important factor for uterine [18,37,38]. In the present study, at the time of death of differentiation, such as decidualization of endometrial the last embryo, P4 concentrations were not basal in all stromal cells or pathological situations. The trend animals and P4 concentrations as high as 84.9 nmol/l towards higher relaxin values in group II was not were measured 45 days after the beginning of abortion. statistically significant. However, an interaction After induction of abortion with a combination of between glucocorticoids and relaxin, as described for aglepristone and cabergoline, P4 concentrations

Fig. 2. Average E2 values after induction of abortion with Alizine (a) Fig. 3. Average P4 values after induction of abortion with Alizine (a) and Alizine + cabergoline (b), before and during abortions. 1, 2 and Alizine + cabergoline (b), before and during abortions. 1, 2 Alizine = 1st and 2nd application of Alizine. Alizine = 1st and 2nd application of Alizine. 894 S. Scha¨fer-Somi et al. / Theriogenology 68 (2007) 889–895 decreased within 48 h after first medication. In a [7] Romagnoli SE, Cela M, Camillo F. Use of prostaglandin F2 previous study [1], P4 concentrations decreased in at alpha for early pregnancy termination in the mismated bitch. Vet Clin North Am Small Anim Pract 1991;21:487–99. least 66.7% of cases, when cabergoline was given at day [8] Romagnoli SE, Camillo F, Cela M, Johnston SD, Grassi F, 30 after mating. The combined medication used in the Ferdeghini M, et al. Clinical use of prostaglandin F2 alpha to present study accelerated the beginning of abortion induce early abortion in bitches: serum progesterone, treatment significantly, a feat that could be explained by the outcome and interval to subsequent oestrus. J Reprod Fertil synergy of the anti-progesterone action and the indirect Suppl 1993;47:425–31. [9] Fieni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for luteolytic effect of cabergoline. pregnancy termination in bitches using prostaglandin F2a.J From the present results, no influence of any Reprod Fertil Suppl 1997;51:245–50. medication on the ovarian secretion of E2 could be [10] Onclin K, Verstegen JP. Practical use of combination of dopa- detected. On average, the course of E2 in both groups mine agonist and synthetic prostaglandin analogue to terminate was similar to that observed in pregnant dogs [39,40] unwanted pregnancy in dogs. J Small Anim Pract 1996;37: 211–6. and cats after induction of abortion with aglepristone [11] Onclin K, Verstegen JP. Comparisons of different combinations [35,36]. of analogues of PGF2 alpha and dopamine agonists for the In conclusion, with both medication schemes, in the termination of pregnancy in dogs. Vet Rec 1999;144:416–9. majority of cases the subsequent cycles were normal [12] Aslan S, Eru¨nal-Maral N, Findik M, Bastan A, Handler J, and fertile. We therefore suggest that the here measured Arbeiter K. Tra¨chtigkeitsabbruch bei der Hu¨ndin durch kombi- nierte Gabe eines PGF2a-Analogons (Alfaprostol oder Clopros- relaxin, E2 and P4 concentrations until day 45 after the tenol) und des Ergolinderivates Cabergolin. Kleintierpraxis end of abortions, were within the bound of normal. In 2001;46:141–8. addition, both schemes were found to be useful for [13] Gobello C, Castex G, Corrada Y, Klima L, de la Sota RL, repeated induction of abortion in bitches. Nevertheless, Rodriguez R. Use of prostaglandins and bromocriptine mesylate after the first application, the combined medication lead for pregnancy termination in bitches. J Am Vet Med Assoc 2002;220:1017–9. to a significant quicker termination of abortion, [14] Fieni F, Tainturier D, Bruyas JF, Badinand F, Berthelot X, Ronsin rendering the combination of aglepristone and cabergo- P, et al. Etude clinique d‘une anti-hormone pour provoquer line an interesting alternative to the sole application of l’avortement chez la chienne: l’aglepristone. Recueil de Med- aglepristone. Considering in addition the different icine Veterinaire 1996;172(7/8):359–67. course of P4 serum concentrations in both groups, [15] Concannon PW, Yeager A, Frank D, Iyampillai A. Termination of pregnancy and induction of premature luteolysis by the we suppose that the interplay of these drugs should be antiprogestagen, mifepristone, in dogs. J Reprod Fertil investigated more closely. 1990;88:99–104. 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Decrease of body temperature after aglepristone treatment in bitches

Y. Corrada a, P. Garc´ıa b, P.E. de la Sota b, M. Huzman a, M.F. Landoni a, C. Gobello a, ∗

a Clinical Department, Faculty of Veterinary Sciences, National University of La Plata, La Plata CC 296, Argentina b Basic Sciences Department, Faculty of Veterinary Sciences, National University of La Plata, La Plata CC 296, Argentina Received 31 July 2004; received in revised form 18 October 2004; accepted 8 November 2004

Abstract

Body temperature responses and the timing of abortions were evaluated in pregnant bitches with the anti-progestin aglepristone. Fifteen purebred and crossbred, 25–45 days pregnant, were included in this study and seven untreated bitches at the same stage of pregnancy served as controls. Treated bitches were administered two applications of aglepristone (10 mg/kg SC) 24 h apart for pregnancy termination. Pregnancy termination was confirmed by ultrasonographic assessment. Body temperature was rectally measured three times a day for 6 days beginning 24 h before treatment or pregnancy diagnosis in the treated and control bitches, respectively. Additionally, serum progesterone concentrations were assessed at time points during the study in the treated bitches. Pregnancy was terminated in 14 treated bitches in a mean ± S.E.M. of 4.3 ± 0.7 days after treatment. Control bitches remained pregnant. In the treated bitches, but not in the controls, body temperature significantly decreased 24 h after the beginning of the treatments (P < 0.01) and then gradually returned to pre-treatment values. Correlation between the day of mean minimum body temperature and the day of pregnancy termination was low (0.07; >0.05). Progesterone did not show significant change throughout the study. Body temperature does not seem to be a suitable variable to clinically monitor the aborting effect of aglepristone. Decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic effects. © 2005 Elsevier B.V. All rights reserved.

Keywords: Bitch; Pregnancy termination; Aglepristone; Body temperature

∗ Corresponding author. Fax: +54 221 425 7980. E-mail addresses: [email protected], [email protected] (C. Gobello).

1. Introduction

Anti-progestins are synthetic steroids which bind with great affinity to progesterone (P4) receptors preventing native P4 from exerting its biological effects (Hoffmann and Schuler, 2000). In dogs, two anti-progestins have been studied mifepristone (RU 486; Concannon et al., 1990; Linde-Forsberg et al., 1992) and aglepristone (RU 534), although only aglepristone is available in the veterinary market of some European and American countries. Aglepristone functions as a true P4 antagonist at the uterus without initially decreasing P4 serum concentrations. It is, therefore, utilized in P4 dependent uterine conditions and diseases. The main clinical indication for anti-progestins is termination of unwanted pregnancy. Aglepristone has shown to efficiently terminate gestation in bitches within 7 days (Fieni et al., 1996, 2001; Galac et al., 2000), being one of the safest options for this purpose. Elevated plasma prolactin concentrations were described within 24 h aglepristone treatment without changing P4 blood concentrations (Fieni et al., 1996; Galac et al., 2000). Prolactin returned to basal concentrations in 2–4 days. Progesterone concentrations declined within 8 and 34 days to basal concentrations due to premature luteolysis (Galac et al., 2000). A decrease of the interestrus intervals by shortening not only of diestrus but also of anestrus was noted in the three studies after aglepristone treatment (Fieni et al., 1996, 2001; Galac et al., 2000). These effects (i.e. prolactin release, premature luteolysis and shortening of the anestrus) of aglepristone may be due to a direct or indirect action of aglepristone on hypothalamic- pituitary axis. Detailed studies in dogs on the effect of P4 receptor antagonist at the hypothalamus–pituitary level are lacking. In a first study in which mifepristone was used for pregnancy termination, body temperature declined 24–48 h after treatment and then returned to normal (Linde-Forsberg et al., 1992). More recently, in aglepristone-treated pregnant bitches, rectal temperature was described to decrease within 48 h after abortion and then to return to normal values (Fieni et al., 1996). Therefore, body temperature monitoring would potentially be a practical tool to follow up anti-progestin effects and to clinically diagnose pregnancy termination. It was, therefore, of interest to describe the body temperature variations in aglepristone-treated pregnant bitches in relation to time of administration and pregnancy termination.

2. Materials and methods

Fifteen 1–13-year-old crossbred and purebred pregnant bitches, 6–28 kg, that were pri- vately owned were treated with two applications of aglepristone (Alizine®, Virbac, France) SC, 10 mg/kg 24 h apart for pregnancy termination. Treatments occurred during the morn- ing hours. The bitches had been bred 25–45 days before and pregnancy confirmed by ultrasonography (Toshiba Core Vision Pro, Japan; Mattoon and Nyland, 1995). During the period of the study, seven bitches with the same characteristics were used as controls. All the bitches were hospitalized during the study, fed commercial dog food and given water ad libitum. The bitches were clinically examined and body temperature was rectally measured three times a day (8 a.m., 4 a.m. and 12 p.m.) for six consecutive days beginning 24 h before Y. Corrada et al. / Animal Reproduction Science 87 (2005) 295–299 297 treatment or pregnancy diagnosis in the treated and control bitches, respectively. Pregnancy termination was clinically diagnosed by the appearance of vaginal discharges, expulsion of fetuses, or typical behavioral changes and immediately confirmed by ultrasonography (Mattoon and Nyland, 1995). Additionally, blood samples by peripheral venepuncture were collected for serum P4 concentration determination before treatment, at the time of ultrasonographic confirmation of pregnancy interruption and 72 h later in the treated bitches. Serum P4 was measured by radioimmunoassay, using a solid phase kit (Coat-A-Count, DPC®, Los Angeles, USA). For this kit, the sensitivity at 95% binding was 0.1 ng/ml and intra-assay CV was 5.7%. Descriptive statistics was performed for days to achieve pregnancy termination and expressed as mean ± S.E.M. Body temperature on the treated and control groups were analyzed by ANOVA for repeated measures and Student–Newman was performed as post- test. A correlation analysis was also performed between the day of mean minimum body temperature and the day of pregnancy termination. The level of significance was set at 0.05.

3. Results

Pregnancy was terminated in 14 of 15 treated bitches either by resorption (n = 8) or abortion (n = 6) in a mean ± S.E.M. of 4.3 ± 0.7 days (range 2–8) after initiation of treatments. As expected, earlier pregnancies terminated mainly by resorption while older ones by abortion. The non-responding bitch was treated with another pharmacological aborting protocol on day 9. Control bitches remained pregnant and whelped normal litters.

Fig. 1. Mean body temperature of 15 pregnant bitches treated with two applications of aglepristone SC (10 mg/kg 24 h apart) for pregnancy termination. Bars on the symbols represent SEM. Asterisks point out signiﬁcantly different (<0.01) values among the days of the treated bitches. (Inset) Mean body temperature of seven control bitches which did not receive any treatment. 298 Y. Corrada et al. / Animal Reproduction Science 87 (2005) 295–299

Fig. 2. Mean serum progesterone of the same treated bitches represented in Fig. 1. Bars on the symbols represent S.E.M.

Body temperature significantly differed between treated and control bitches (Fig. 1, inset) and varied throughout the study in the treated bitches, consistently decreasing 24 h after the beginning of the treatments (P < 0.01) and returning gradually to pre-treatment values (Fig. 1). Correlation between the day of averaged minimum body temperature and the day of pregnancy termination was low (0.07) and non-significant. Progesterone did not show significant change in the treated bitches, although a tendency for a decrease was evident as expected in this phase of pregnancy (Fig. 2).

4. Discussion and conclusion

Aglepristone successfully terminated pregnancy in most of the treated bitches. In line with a previous report, in the present trial efficacy was slightly less than 100% (Fieni et al., 1996). Gestation interruption occurred in the presence of P4 concentrations that were typical of those during pregnancy confirming the local uterine effect of the receptor blocker. Progesterone is a thermogenic hormone. Its action at central nervous system is responsible for decreases in body temperature (Guyton and Hall, 1997). Therefore, body temperature variations in the present study could not be attributed to a peripheral decrease of serum P4. In contrast to a former study (Fieni et al., 1996), in the present study, no chronological relationship was found between pregnancy termination and variation on body temperature. Temperature consistently decreased 24 h after treatment in all the animals, independently of the time at which pregnancy termination occurred. The decline in body temperature, even in the bitch in which the treatment failed, further confirmed this observation. A similar Y. Corrada et al. / Animal Reproduction Science 87 (2005) 295–299 299 temperature pattern was described in three bitches after mifepristone administration in an earlier report (Linde-Forsberg et al., 1992). Thus, decreased body temperature after treatment could represent additional evidence of anti-progestin effects on the hypothalamus and other central nervous system centers. The abrupt decrease in body temperature may be explained by occupation of central P4 receptors by the antagonist, mimicking a sudden decline in P4 concentrations. Interestingly, plasma prolactin has shown an opposite pattern of variation after aglepristone administration (Fieni et al., 2001; Galac et al., 2000). Prolactin is known to increase in response to P4 deprivation in this species (Gobello et al., 2001). Thus, prolactin may promote similar, although opposite, actions of anti-progestins at hypothalamus. It is concluded that body temperature does not seem to be a reliable variable to clinically monitor aglepristone aborting effect because no relationship between this variable and pregnancy termination time was determined in the present study. Additionally, decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic and other central nervous system center effects.

Acknowledgement

The authors would like to thank Virbac, France for Alizine® supply.

References

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