Toxicology, 5 (1975) 97--101 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
THE EFFECTS OF PROSTAGLANDIN E2 ON PREGNANCY AND EMBRYONIC DEVELOPMENT IN MICE
T.V.N. PERSAUD Teratology Research Laboratory, Departmenl of Anatomy, The University of Manitoba, Bannatyne and Emily, Winnipeg, Manitoba (Canada R3E OW3)
(Received March 18th, 1975) (Accepted June 3rd, 1975)
SUMMARY The prostaglandins comprise an important and unique group of naturally occurring biological regulators. These substances are currently being widely used for the induction of labor and termination of pregnancy. The effects of prostaglandin E2 (PGE2) on pregnancy and embryonic development were investigated in Swiss-Webster mice. PGE 2 induced a high incidence of abnormal offspring in mice when administered subcutaneously on either day 9, 12 or 15 of gestation. In addition, fetal growth was significantly affected following treatment with the prostaglandin on either the 12th or 15th day of gestation.
INTRODUCTION The prostaglandins represent a unique group of naturally occurring biological regulators which are currently being widely used for the induction of labor and as an abortifacient in pregnant women. In view of the thalidomide disaster and the known teratogenic hazards of certain drugs, the possible teratogenic risks of prostaglandins need to be evaluated [1]. The anti-fertility effects of PGE2 in rats, hamsters [2,3] and rabbits [2] ; of PGE 1 in rats and hamsters [3] ; and of PGE2, PGF2~ and PGE1 in mice [4] have been reported. There is, however, no evidence for a teratogenic action by these substances [1]. The present communication describes the effects of PGE 2 on pregnancy and embryonic development in mice. Abbreviation: PG, prostaglandin.
97
MATERIALS AND METHODS
Female Swiss-Webster mice 10--12 weeks of age and weighing 32--35 g were caged overnight with fertile males of the same strain, using one male for two females. At 9 a.m. the following morning mating was determined by the presence of a vaginal plug or occlusion of the vaginal orifice; this was considered to be the first day of pregnancy. The total number of female animals mated in this manner was 130, and 71 of 86 (> 82%) animals showing vaginal plugs became pregnant. 71 pregnant animals in groups of 3 or 4 were kept in plastic cages with straight wire lids, and maintained under controlled environmental conditions (12-h light--dark sequences, temperature 72°F, 50% relative humidity). Purina mouse chow and water were provided ad lib. PGE2 (25 gg) was dissolved in physiological saline and administered subcutaneously to each of three groups of mice once on either the 9th, 12th or 15th day of gestation. Each treated group and the corresponding control group consisted of 11--13 pregnant animals (see Table I). All control animals were treated with the solvent only. The mothers were carefully observed for weight loss and vaginal bleeding following treatment. On day 20 of gestation all animals were killed. The fetuses were weighed and examined for external malformations. The number of resorption sites were also recorded. Most fetuses were fixed in Bouin's fluid. Free-hand razor blade sections of the fetuses were made and examined for visceral malformations [5]. In addition, fetuses from each treated group were examined for skeletal abnormalities after being fixed in ethyl alcohol, cleared and stained with alizarin red [6]. TABLE I P R O S T A G L A N D I N E 2 T R E A T M E N T D U R I N G P R E G N A N C Y IN MICE Number o f pregnant animals
Treatment
12
25
11
Controls
12
25
12
Controls
13
25
11
Controls
a b c d
(pg)
Implantations Resorptionsa (%) Day
9
12
15
F e t u s e s recovered Alive
Dead
Abnormal fetuses b (%)
53 (40.8) d
142
12 (8.5) c
130
0
124
5 (4.0)
117
2
2 (1.7)
138
3 (2.2) c
133
2
58 (43) d
127
5 (3.9)
121
1
126
3 (2.4) c
121
2
120
7 (5.8)
108
5
P e r c e n t a g e based o n n u m b e r o f i m p l a n t a t i o n s . Percentage based on total n u m b e r o f fetuses recovered. NS (Significance o f d i f f e r e n c e f r o m c o n t r o l was evaluated by X2-test). p ~ 0.001.
98
0 26(21.1) d 1 (0.9)
~D
Abnormal fetuses /fetuses recovered
53/130 2/119
581135 0/122
26/123 1/113
Day of treatment
9 Experimental Control
Experimental 12 C o n t r o l
Experimental 15 C o n t r o l
3 0
2 0
8 0
Stunting
6 0
1 0
0 0
8 0
9 0
13 2
Meromelia Hydrocephalus
7 1
28 0
31 0
Microphthalmia
1 0
0 0
1 0
Spina bifida
P R O S T A G L A N D IN E 2 I N D U C E D C O N G E N I T A L M A L F O R M A T I O N S IN MICE
T A B L E II
0 0
12 0
0 0
Polycystic kidney
0 0
6 0
0 0
Absence of urinary bladder
0 0
0 0
Omphalocele
RESULTS The results are s u m m a r i z e d in T a b l e I. T h e r e were n o m a t e r n a l d e a t h s and t h e r e were no signs o f vaginal bleeding following t r e a t m e n t . T h e m e a n b o d y weights indicated n o d i f f e r e n c e b e t w e e n t r e a t e d a n d c o n t r o l animals w i t h r e s p e c t to weight gain during the e x p e r i m e n t a l p e r i o d . T r e a t m e n t o f p r e g n a n t m i c e with a single dose o f PGE2 (25 pg), administered s u b c u t a n e o u s l y on the d i f f e r e n t g e s t a t i o n a l d a y s d u r i n g e m b r y o n i c d e v e l o p m e n t , did n o t significantly increase the i n c i d e n c e o f e m b r y o n i c d e a t h s and fetal r e s o r p t i o n s , c o m p a r e d t o the c o r r e s p o n d i n g c o n t r o l s . T h e n u m b e r o f viable o f f s p r i n g r e c o v e r e d near t e r m was n o t a f f e c t e d b y t r e a t m e n t . In all t r e a t m e n t groups, t h e incidence o f a b n o r m a l fetuses was significantly g r e a t e r c o m p a r e d to the c o r r e s p o n d i n g c o n t r o l s (Table I). A survey o f m a j o r d e v e l o p m e n t a l d e f e c t s i n d u c e d b y t h e p r o s t a g l a n d i n is given in Table II. Major skeletal m a l f o r m a t i o n s were n o t p r e s e n t in a n y o f t h e alizarin-stained fetuses. H o w e v e r , a relatively high incidence o f m i n o r skeletal v a r i a t i o n s was d e t e c t e d in b o t h c o n t r o l fetuses and fetuses t r e a t e d with t h e p r o s t a g l a n d i n . T h e s e i n c l u d e d i n c o m p l e t e s t e r n e b r a e , missing phalanges, r u d i m e n t a r y ribs, a n d p o o r ossification o f the skull, v e r t e b r a e and ribs. T h e i n t r a u t e r i n e g r o w t h o f t h e o f f s p r i n g was a f f e c t e d f o l l o w i n g t r e a t m e n t w i t h PGE 2 on days 12 and 15, b u t n o t on d a y 9 o f gestation. In t h e f o r m e r cases, t h e m e a n fetal b o d y weight a n d m e a n c r o w n - r u m p length o f p r o s t a gland±n-treated fetuses were significantly d i f f e r e n t f r o m t h o s e o f t h e c o n t r o l s (Table III).
TABLE III EFFECT OF PROSTAGLANDIN E 2 ON FETAL GROW'Ytt Gestational day of treatment
Fetal weight (in g; Mean -+ SEM)
Fetal length (in mm; Mean -+ SEM)
9 Experimental Control
0.998 ± 0.021 a 0.960 ± 0.035
19.272 -+ 0.185 a 19.508 ± 0.354
12 Experimental Control
1.111 ± 0.017 b 1.222 -+ 0.027
20.818 + 0 . 2 0 6 b 21.871 + 0.370
15 Experimental Control
0.895 ± 0.029 b 1.089 ± 0.034
20.031 -+ 0.293 b 21.377 +- 0.255
Differences between the values of the control group and the prostaglandin-treated groups were analyzed by means of the Student " t " test. a NS. b p~: 0.01.
I00
DISCUSSION In contrast to previous findings in the rat [7] the results of the present investigation showed that PGE 2 is highly teratogenic in mice. However, it was not evident from the results whether the embryo is more susceptible to teratogenesis following treatment on any particular gestational day. At the dose level used (25 pg) the incidence of fetal resorptions was not significantly increased, but fetuses surviving to term showed a high incidence of developmental defects. The malformations detected in the offspring were of several different morphological types and are often seen following treatment with other teratogenic agents [8]. It would appear from our data that PGE2 is n o t particularly lethal to the mouse conceptus following treatment at critical developmental periods, since the incidence of fetal resorptions seen at term was not significantly different from the controls, an observation previously reported by Marley [4]. It is difficult to explain the lack of effect on the intrauterine growth of the fetus after treatment on gestational day 9; the mean crown-rump length and mean fetal weight showed no significant differences from the corresponding controls. However, treatment on the other gestational days did cause a marked decrease in both fetal length and weight. Nevertheless, fetuses surviving to term following treatment on day 9 of gestation, as well as on day 12 or 15, showed a significantly high incidence of congenital abnormalities. The mode of action of PGE2 in the pregnant mouse is not known. Teratogenic effects may be produced by several different mechanisms involving the mother, the fetus or the feto-placental unit. Previous work from our laboratory has shown that PGE2 and/or its metabolites readily cross the mouse placenta. Significant levels of these substances have been detected in the conceptuses [9]. In addition, pre-treatment of pregnant mice with PGE 2 significantly decreased (more than 50%) the uptake of tritiated thymidine in the embryos [10]. Thus, a direct action of the prostaglandin on the developing conceptus may play an important part in accounting for the adverse effects produced by this substance in the pregnant mouse. These results in mice cannot be applied directly to human pregnancies, but suggest grounds for caution in using PGE 2 during early pregnancy. Further studies with PGE 2 in several species following administration by different routes are necessary in order to assess possible teratogenic properties fully. ACKNOWLEDGEMENTS This work was supported by a grant from the Medical Research Council of Canada. I am grateful to Mr. Kazuo Sano, Ono Pharmaceutical Co. Ltd., Osaka, Japan for supplying the prostaglandin. I wish to thank Dr. K.L. Moore for valuable suggestions, Miss M. Wiebe and Mr. B. Sam for their excellent technical assistance, and Miss R. Biedron for typing the manuscript.
101
THE EFFECTS OF PROSTAGLANDIN E2 ON PREGNANCY AND EMBRYONIC DEVELOPMENT IN MICE
T.V.N. PERSAUD Teratology Research Laboratory, Departmenl of Anatomy, The University of Manitoba, Bannatyne and Emily, Winnipeg, Manitoba (Canada R3E OW3)
(Received March 18th, 1975) (Accepted June 3rd, 1975)
SUMMARY The prostaglandins comprise an important and unique group of naturally occurring biological regulators. These substances are currently being widely used for the induction of labor and termination of pregnancy. The effects of prostaglandin E2 (PGE2) on pregnancy and embryonic development were investigated in Swiss-Webster mice. PGE 2 induced a high incidence of abnormal offspring in mice when administered subcutaneously on either day 9, 12 or 15 of gestation. In addition, fetal growth was significantly affected following treatment with the prostaglandin on either the 12th or 15th day of gestation.
INTRODUCTION The prostaglandins represent a unique group of naturally occurring biological regulators which are currently being widely used for the induction of labor and as an abortifacient in pregnant women. In view of the thalidomide disaster and the known teratogenic hazards of certain drugs, the possible teratogenic risks of prostaglandins need to be evaluated [1]. The anti-fertility effects of PGE2 in rats, hamsters [2,3] and rabbits [2] ; of PGE 1 in rats and hamsters [3] ; and of PGE2, PGF2~ and PGE1 in mice [4] have been reported. There is, however, no evidence for a teratogenic action by these substances [1]. The present communication describes the effects of PGE 2 on pregnancy and embryonic development in mice. Abbreviation: PG, prostaglandin.
97
MATERIALS AND METHODS
Female Swiss-Webster mice 10--12 weeks of age and weighing 32--35 g were caged overnight with fertile males of the same strain, using one male for two females. At 9 a.m. the following morning mating was determined by the presence of a vaginal plug or occlusion of the vaginal orifice; this was considered to be the first day of pregnancy. The total number of female animals mated in this manner was 130, and 71 of 86 (> 82%) animals showing vaginal plugs became pregnant. 71 pregnant animals in groups of 3 or 4 were kept in plastic cages with straight wire lids, and maintained under controlled environmental conditions (12-h light--dark sequences, temperature 72°F, 50% relative humidity). Purina mouse chow and water were provided ad lib. PGE2 (25 gg) was dissolved in physiological saline and administered subcutaneously to each of three groups of mice once on either the 9th, 12th or 15th day of gestation. Each treated group and the corresponding control group consisted of 11--13 pregnant animals (see Table I). All control animals were treated with the solvent only. The mothers were carefully observed for weight loss and vaginal bleeding following treatment. On day 20 of gestation all animals were killed. The fetuses were weighed and examined for external malformations. The number of resorption sites were also recorded. Most fetuses were fixed in Bouin's fluid. Free-hand razor blade sections of the fetuses were made and examined for visceral malformations [5]. In addition, fetuses from each treated group were examined for skeletal abnormalities after being fixed in ethyl alcohol, cleared and stained with alizarin red [6]. TABLE I P R O S T A G L A N D I N E 2 T R E A T M E N T D U R I N G P R E G N A N C Y IN MICE Number o f pregnant animals
Treatment
12
25
11
Controls
12
25
12
Controls
13
25
11
Controls
a b c d
(pg)
Implantations Resorptionsa (%) Day
9
12
15
F e t u s e s recovered Alive
Dead
Abnormal fetuses b (%)
53 (40.8) d
142
12 (8.5) c
130
0
124
5 (4.0)
117
2
2 (1.7)
138
3 (2.2) c
133
2
58 (43) d
127
5 (3.9)
121
1
126
3 (2.4) c
121
2
120
7 (5.8)
108
5
P e r c e n t a g e based o n n u m b e r o f i m p l a n t a t i o n s . Percentage based on total n u m b e r o f fetuses recovered. NS (Significance o f d i f f e r e n c e f r o m c o n t r o l was evaluated by X2-test). p ~ 0.001.
98
0 26(21.1) d 1 (0.9)
~D
Abnormal fetuses /fetuses recovered
53/130 2/119
581135 0/122
26/123 1/113
Day of treatment
9 Experimental Control
Experimental 12 C o n t r o l
Experimental 15 C o n t r o l
3 0
2 0
8 0
Stunting
6 0
1 0
0 0
8 0
9 0
13 2
Meromelia Hydrocephalus
7 1
28 0
31 0
Microphthalmia
1 0
0 0
1 0
Spina bifida
P R O S T A G L A N D IN E 2 I N D U C E D C O N G E N I T A L M A L F O R M A T I O N S IN MICE
T A B L E II
0 0
12 0
0 0
Polycystic kidney
0 0
6 0
0 0
Absence of urinary bladder
0 0
0 0
Omphalocele
RESULTS The results are s u m m a r i z e d in T a b l e I. T h e r e were n o m a t e r n a l d e a t h s and t h e r e were no signs o f vaginal bleeding following t r e a t m e n t . T h e m e a n b o d y weights indicated n o d i f f e r e n c e b e t w e e n t r e a t e d a n d c o n t r o l animals w i t h r e s p e c t to weight gain during the e x p e r i m e n t a l p e r i o d . T r e a t m e n t o f p r e g n a n t m i c e with a single dose o f PGE2 (25 pg), administered s u b c u t a n e o u s l y on the d i f f e r e n t g e s t a t i o n a l d a y s d u r i n g e m b r y o n i c d e v e l o p m e n t , did n o t significantly increase the i n c i d e n c e o f e m b r y o n i c d e a t h s and fetal r e s o r p t i o n s , c o m p a r e d t o the c o r r e s p o n d i n g c o n t r o l s . T h e n u m b e r o f viable o f f s p r i n g r e c o v e r e d near t e r m was n o t a f f e c t e d b y t r e a t m e n t . In all t r e a t m e n t groups, t h e incidence o f a b n o r m a l fetuses was significantly g r e a t e r c o m p a r e d to the c o r r e s p o n d i n g c o n t r o l s (Table I). A survey o f m a j o r d e v e l o p m e n t a l d e f e c t s i n d u c e d b y t h e p r o s t a g l a n d i n is given in Table II. Major skeletal m a l f o r m a t i o n s were n o t p r e s e n t in a n y o f t h e alizarin-stained fetuses. H o w e v e r , a relatively high incidence o f m i n o r skeletal v a r i a t i o n s was d e t e c t e d in b o t h c o n t r o l fetuses and fetuses t r e a t e d with t h e p r o s t a g l a n d i n . T h e s e i n c l u d e d i n c o m p l e t e s t e r n e b r a e , missing phalanges, r u d i m e n t a r y ribs, a n d p o o r ossification o f the skull, v e r t e b r a e and ribs. T h e i n t r a u t e r i n e g r o w t h o f t h e o f f s p r i n g was a f f e c t e d f o l l o w i n g t r e a t m e n t w i t h PGE 2 on days 12 and 15, b u t n o t on d a y 9 o f gestation. In t h e f o r m e r cases, t h e m e a n fetal b o d y weight a n d m e a n c r o w n - r u m p length o f p r o s t a gland±n-treated fetuses were significantly d i f f e r e n t f r o m t h o s e o f t h e c o n t r o l s (Table III).
TABLE III EFFECT OF PROSTAGLANDIN E 2 ON FETAL GROW'Ytt Gestational day of treatment
Fetal weight (in g; Mean -+ SEM)
Fetal length (in mm; Mean -+ SEM)
9 Experimental Control
0.998 ± 0.021 a 0.960 ± 0.035
19.272 -+ 0.185 a 19.508 ± 0.354
12 Experimental Control
1.111 ± 0.017 b 1.222 -+ 0.027
20.818 + 0 . 2 0 6 b 21.871 + 0.370
15 Experimental Control
0.895 ± 0.029 b 1.089 ± 0.034
20.031 -+ 0.293 b 21.377 +- 0.255
Differences between the values of the control group and the prostaglandin-treated groups were analyzed by means of the Student " t " test. a NS. b p~: 0.01.
I00
DISCUSSION In contrast to previous findings in the rat [7] the results of the present investigation showed that PGE 2 is highly teratogenic in mice. However, it was not evident from the results whether the embryo is more susceptible to teratogenesis following treatment on any particular gestational day. At the dose level used (25 pg) the incidence of fetal resorptions was not significantly increased, but fetuses surviving to term showed a high incidence of developmental defects. The malformations detected in the offspring were of several different morphological types and are often seen following treatment with other teratogenic agents [8]. It would appear from our data that PGE2 is n o t particularly lethal to the mouse conceptus following treatment at critical developmental periods, since the incidence of fetal resorptions seen at term was not significantly different from the controls, an observation previously reported by Marley [4]. It is difficult to explain the lack of effect on the intrauterine growth of the fetus after treatment on gestational day 9; the mean crown-rump length and mean fetal weight showed no significant differences from the corresponding controls. However, treatment on the other gestational days did cause a marked decrease in both fetal length and weight. Nevertheless, fetuses surviving to term following treatment on day 9 of gestation, as well as on day 12 or 15, showed a significantly high incidence of congenital abnormalities. The mode of action of PGE2 in the pregnant mouse is not known. Teratogenic effects may be produced by several different mechanisms involving the mother, the fetus or the feto-placental unit. Previous work from our laboratory has shown that PGE2 and/or its metabolites readily cross the mouse placenta. Significant levels of these substances have been detected in the conceptuses [9]. In addition, pre-treatment of pregnant mice with PGE 2 significantly decreased (more than 50%) the uptake of tritiated thymidine in the embryos [10]. Thus, a direct action of the prostaglandin on the developing conceptus may play an important part in accounting for the adverse effects produced by this substance in the pregnant mouse. These results in mice cannot be applied directly to human pregnancies, but suggest grounds for caution in using PGE 2 during early pregnancy. Further studies with PGE 2 in several species following administration by different routes are necessary in order to assess possible teratogenic properties fully. ACKNOWLEDGEMENTS This work was supported by a grant from the Medical Research Council of Canada. I am grateful to Mr. Kazuo Sano, Ono Pharmaceutical Co. Ltd., Osaka, Japan for supplying the prostaglandin. I wish to thank Dr. K.L. Moore for valuable suggestions, Miss M. Wiebe and Mr. B. Sam for their excellent technical assistance, and Miss R. Biedron for typing the manuscript.
101
