Induction of preterm labor in the rat by antiprogesterone ARPAD BELA St. Louis,
I. A.
CSAPO RESCH
Missouri,
and
Szeged,
Hungary
The validity of a new concept, predicting that preterm labor can be induced without oxytocic stimuration by a regulatory imbalance, generated by antiprogesterone (A-P)-provoked P withdrawal (Pw), has been examined in the rat model. At day 19 of pregnancy a single oral dose of the steroidal A-P isoxazol, which inhibits P synthesis, significantly reduced uterine P levels, increased uterine ezstradiol and prostaglandin F levels, and induced preterm labor. This precocious regulatory imbalance and preterm labor were prevented, by blocking the A-P-induced Pw with P treatment. The regulatory imbalance which triggered the onset of spontaneous labor in the control animals was similar, but it occurred at term rather than before term. The potential of this method in improving the management of medically indicated induction of preterm labor is discussed. (AM. J. OBSTET. GYNECOI..
134:823,
1979.)
continue to debate the proper management of the termination of pregnancy when it is medically indicated before term. Modern textbooks of obstetrics’ recommend that labor be induced with oxytocic drugs and advise that cesarean section should be considered only as a last resort, when pharmacologic stimulation of the uterus has failed. However, it is emphasized that the less advanced the pregnancy, the more unpredictable the outcome of uterine stimulation and that beyond permissible limits and during prolonged application uterine stimulants become dangerous to both mother and fetus. Thus, if cesarean section eventually has to be performed after induction failure, new risks are added to those which already existed before induction. Therefore, intensive research work is recommended’ for the development of “safe and more effective methods of inducing labor, to reduce the need for cesarean section and the major maternal and fetal hazards created by induction with oxytocic drugs.” The persistent uncertainties regarding the proper conduct of a routine clinical procedure reflect the failPERINATOLOGISTS
From the Departments of Obstetrics and Gynecology, Washington University Schaol of Medicine and Szeged Universzty Medical School. Su#ortt*d Department
by the Agency for International Development, of State, Contract No. AIDlpha-C-I 193.
Reprint requests: Dr. Arpad I. Csapo, Department Obstetrics and Gynecology, 4911 Barnes Hospital St. Louii, Missouri 6?110. 000%9378/791150823+05$00.50/0~
1979The
C.V.
Mosby
of Plaza,
Co.
ure of academic obstetrics in defining: (1) the basic regulatory mechanism of the pregnant human uterus, (2) the endogenous changes in regulatory balance during third-trimester pregnancy, and (3) the diagnostic tests which quantitate them. Since the controlling mechanism of pregnancy is complex and since in the human the uterus is not accessible for sequential sampling, nor is the uterine vein, as it is in the sheep or the goat,2 an early breakthrough in the exposure of this mechanism is not within reach. Therefore, new concepts have to be developed in animal models for the safe and reliable elective termination of pregnancy in preparation for eventual clinical trials. This article describes such a concept and current experiments which validate it. Despite the controversy regarding the role of progesterone (P) in the initiation of spontaneous labor at term,3 it is informative to examine the effect of pharmacologically induced P withdrawal (Pw) on the unstimulated preterm uterus. A great variety of studies, in both laboratory animals and patients, indicate that pregnancy cannot withstand Pw of a critical degree.3 This is probably so because throughout gestation the uterus is equipped with an endogenous stimulatory mechanism which triggers uterine activity whenever Pw is excessive. The present method of inducing Pw has been studied earlier in the rat, monkey, and sheep,4 with use of synthetic steroids: androstano[2,3-&Jisoxazols. These anti-Ps (A-Ps) predictably terminate mid823
824
Csapo and Resch
Table I. The effect of isoxazol of labor and fetal weight
without
and with P treatment
Animal’s treatment
No. 12 26 9
Vehicle Isoxazol Isoxazol plus
I,iUU size only
on the time ot onset
onsut
10.7 c 0.7 9.7 2 0.4 11.1 -+ 0.7
if lab,(hr.)
29.1 + 2.2 q (J f 2.2” 3:s: I It 1. I
P
All values are mean5 ? standard error. Dose of oral isoxazol = 20 mg and that of intramuscular calculated from 0 hour on day 2 I, *Significantly different from the control values at the level of P < 0.00 1.
Table
II. The effect of isoxazol
on uterine
tissue P, E,, and PGF levels Time after
0 hr. (h*)
Treatment Progesterone
P = 5 mg. The oncct ol‘labor is
treatment
12 //I. (hl
24 hr.
48 /I?-.
(8)
(-5)
42 i- 3:
30 + 2t
15 t 2$
27 t 49”
22 + 2$’
Drlirv?? (9)
f’o,l pnrtum 131
(nglg)
Vehicle
14-
If
14 t 49
17 i- I$
I6 i: YQ
382 k 33t
86 -+ 133
Delivered
290
48
144 * 11$
105 t 91
67 + 8 Isoxazol Esrradeol
Delivered
(Pglg)
Vehicle
207
+ 26
232
t
18
202
+ 18
464
t 4481
493
2 603
273 -t 22
Isoxazol Prostagkdin
k 29
2 lO$
F (nglg)
Vehicle
482
10
95 2 6t
50 t
20
68 -t 7
Isoxazol
102 5 91
135 2 13SY
Delivered
98 *
lit
40
rt: 10
AlI values are means ? standard errors. For the nine isoxazole plus P-treated rats at 48 hours: P = 71 = 7 ng/gm. Ez = 275 2 39 pgigm, and PGF = 89 f 4 ng/gm. *Number of control and isoxazol-treated rats assayed for uterine I’, Ez, and PGF ai the specified rimea tDifference
statistically
significant
compared
to pretreatment
fDifference statistically significant compared to pretreatment ODifference statistically significant compared to pretreatment “Difference BDifference
statisticaly statistically
significant significant
compared compared
to control to control
value
at time
0 (p < 0.05).
value at time 0 (p i 0.001). value at time 0 (p < 0.01).
value (p < 0.05). value (p < 0.01).
trimester pregnancy, by blocking P synthesis through the inhibition of 3P-hydroxysteroid dehydrogenase. Sequential radioimmunoassays (RIAs) in the uterine tissues of 10 day pregnant rats showed that a single oral dose of isoxazol, which predictably terminates pregnancy, significantly reduces P and increases estradiol (E2) and prostaglandin F (PGF) levels.” If the premise were verified that in preterm rats a similar regulatory imbalance and parturition are triggered by this same steroidal A-P and indeed if it could be shown that this induced imbalance is similar to that which precedes spontaneous labor at term, a new therapeutic principle could be exposed, since induction with A-P does not demand exogenous oxytocic stimulation. This contention. that it is the isoxazol-induced Pw which provokes
the precocious regulatory imbalance and parturition, and not some unrecognized side effects of A-P, could be substantiated by evidence that concomitant treatment with P prevents the action of isoxazol.
Material and methods During March, 1978, pregnant Sprague-Dawley rats were palpated at the laboratory on day 19 of pregnancy, to exclude animals with low litter size and thus reduce variations in the onset of labor which might be caused by season and litteg size. After this selection 95 animals remained, from which 3’7 were used as controls, 40 were treated orally with 20 mg isoxazol, and 18 were given 5 mg P in addition to isoxazol. Throughout the study, each rat was kept in an individual cage at a
Volume Number
Induction of preterm labor by antiprogesterone
134 7
I
VEHICLE
ISOXAZOL
Labor
t
21 DAIS
OF
PREGNANCY
22
ISOXAZOL
21 DAIS
No
Lb
t
R
20
+ PROGESTERONE
825
22
OF PREGNANCY
P
20 DAYS
OF
21 PREGNANCY
22
Fig. 1. The effect
of isoxazol on uterine tissue P, EP, and PGF levels in preterm rats. At 9 PM on day 1’3 uterine tissue was collected from six animals, and 31 rats subsequently received vehicle, 26 isoxazol (20 mg) and nine isoxazol plus P (20 f 5 mg). At different times after treatment uterine tissue was collected from each group as indicated by symbols. Numbers over symbols describe the numbers of P, EB, and PGF assays in duplicate. Note the nature and timing of the regulatory imbalance preceding isoxazol-induced preterm and normal term labor and the effect of P in preventing the isoxazol action.
constant temperature (22” C), exposed to 14 hours of light and 10 hours of darkness, and fed a diet of Purina chow. The experiment began at 9 PM on day 19 of pregnancy, when the control animals weighed 363 +- 8 grams and the isoxazol-treated rats weighed 350 * 6 grams. As determined at the end of the study, the litter size of the three groups of animals was similar (Table I). The control animals were treated orally with the vehicle (2 ml 1% aqueous gum tragacanth) while the isoxazol and the isoxazol plus P rats received 20 mg isoxazol suspended in the same vehicle. The P was injected intramuscularly (in oil), in a single 5 mg dose, at the same time as the isoxazol. The animals, were observed, then killed after delivery for further workup. Exceptions were six animals, autopsied before treatment at day 19 of pregnancy, and 19 control animals, 14 isoxazol-treated, and nine isoxazol plus P-treated rats killed at different times after treatment, but before parturition, as described in Table II. The remaining 12 control, 26 isoxazoltreated, and nine isoxazol plus P-treated rats were allowed to deliver spontaneously (Tables I and II). With midnight of d,3y 20 taken as time 0, the time of delivery of the first newborn animal in each mother defined the onset of labor. The weights of the fetuses and placentas were measured, to assess the effect of isoxazol on the birth weight of the newborn animals and placentas. In
nine control and nine isoxazol-treated animals uterine tissue was collected at the completion of delivery, while in three control and three isoxazol-treated rats tissue collection was delayed for -3 hours post partum. The uterine tissues were stored at -20” C before measurement of P, EP, and PGF, as described previously.4 These assays were performed to determine whether the isoxazol-induced regulatory imbalance, preceding the onset of preterm labor, (1) is distinct, (2) is similar to that which precedes spontaneous labor at term, and (3) is prevented from occurring by P treatment. The data were analyzed for statistically significant differences using Student’s t test.
Results Table I shows that 12 control animals, allowed to deliver spontaneously, expelled the first fetus at 29.1 L 2.2 hours (mean 2 standard error) after 0 time (e.g., around 5 AM of day 22 of pregnancy). The weight of these term newborn animals was 6.1 t 0.1 grams. In contrast, the 26 isoxazol-treated rats delivered the first fetus at 9.0 f. 0.4 hours (P consequent regulatory imbalanc-e 1” onipdng pretcrnl labor. During and after delivery the previousl\ high b:? and PGF levels decreased precipitate]\ in both rhc controt and isoxaLol-treated rats but the alread! low I’ levels did not. ‘I’hus, the regulatory changes preceding the onset of labor were similar in the control and isoxazol-treated rats, but they occurred significanti) earlier in the isoxazol-treated animals. ‘l’he precocious onset in the isoxazol-treated rats resulted iI1 the birth of living newborn animals with lower than normal lhesr c.orresponded, IIO\~C\CT. \\ith theit weights: shorter gestational ages. (:urrent studies are determining the long-term survival I-ate. weight gain. maturation, and reproductive performance of rhc isoxazoltreated newborn animals. A prediction of the “seesaw” theory’ that the ~ntiogenous stimulatory mechanism the pregnant uterus can be activated and thus labor can be induced before term by mere Pw has apparently been validated b) the present experiments. The contention that this action was due to Pw, and not to some side effect ol’treatment. xas unequivocally established by the demonstration I hat protective treatment Faith P prevented the isosa& induced precocious PM, r11e cotrsequenr regulaCor\ in)balance, and preterm deliver!. 'l‘hris. it appears that the unpredictabilit)of the outcome of preterm itlduction of labor with ox!-to& drugs might be resolved I,> developing neb’ concepts and procedures tar activating the normal pregnant reti.actorv uterus withoul forced oxytocic stimulation. The urgency of turthel- work is reflected by reliable clinical evidence that at 32 to 35 weeks of pregnancy even the combined procedures of rupturing the membranes and oxytocin stimulation have failed to provoke labor during 24 hours in 83% of the cases.” Furthermore, of 1.000 patients, 93% who were 3 days before their expected days of confinement were delivered during the first attempt at oxytocin in-
Volume Number
Induction of preterm labor by antiprogesterone
134 7
duction, while the remaining 7% who were 7 days before their expected days of confinement required repeated inductton. Of course, forced induction by
REFERENCES 1. Danforth, D. N.: Obstetrics and Gynecology, York, 1977, !
I. A.
CSAPO RESCH
Missouri,
and
Szeged,
Hungary
The validity of a new concept, predicting that preterm labor can be induced without oxytocic stimuration by a regulatory imbalance, generated by antiprogesterone (A-P)-provoked P withdrawal (Pw), has been examined in the rat model. At day 19 of pregnancy a single oral dose of the steroidal A-P isoxazol, which inhibits P synthesis, significantly reduced uterine P levels, increased uterine ezstradiol and prostaglandin F levels, and induced preterm labor. This precocious regulatory imbalance and preterm labor were prevented, by blocking the A-P-induced Pw with P treatment. The regulatory imbalance which triggered the onset of spontaneous labor in the control animals was similar, but it occurred at term rather than before term. The potential of this method in improving the management of medically indicated induction of preterm labor is discussed. (AM. J. OBSTET. GYNECOI..
134:823,
1979.)
continue to debate the proper management of the termination of pregnancy when it is medically indicated before term. Modern textbooks of obstetrics’ recommend that labor be induced with oxytocic drugs and advise that cesarean section should be considered only as a last resort, when pharmacologic stimulation of the uterus has failed. However, it is emphasized that the less advanced the pregnancy, the more unpredictable the outcome of uterine stimulation and that beyond permissible limits and during prolonged application uterine stimulants become dangerous to both mother and fetus. Thus, if cesarean section eventually has to be performed after induction failure, new risks are added to those which already existed before induction. Therefore, intensive research work is recommended’ for the development of “safe and more effective methods of inducing labor, to reduce the need for cesarean section and the major maternal and fetal hazards created by induction with oxytocic drugs.” The persistent uncertainties regarding the proper conduct of a routine clinical procedure reflect the failPERINATOLOGISTS
From the Departments of Obstetrics and Gynecology, Washington University Schaol of Medicine and Szeged Universzty Medical School. Su#ortt*d Department
by the Agency for International Development, of State, Contract No. AIDlpha-C-I 193.
Reprint requests: Dr. Arpad I. Csapo, Department Obstetrics and Gynecology, 4911 Barnes Hospital St. Louii, Missouri 6?110. 000%9378/791150823+05$00.50/0~
1979The
C.V.
Mosby
of Plaza,
Co.
ure of academic obstetrics in defining: (1) the basic regulatory mechanism of the pregnant human uterus, (2) the endogenous changes in regulatory balance during third-trimester pregnancy, and (3) the diagnostic tests which quantitate them. Since the controlling mechanism of pregnancy is complex and since in the human the uterus is not accessible for sequential sampling, nor is the uterine vein, as it is in the sheep or the goat,2 an early breakthrough in the exposure of this mechanism is not within reach. Therefore, new concepts have to be developed in animal models for the safe and reliable elective termination of pregnancy in preparation for eventual clinical trials. This article describes such a concept and current experiments which validate it. Despite the controversy regarding the role of progesterone (P) in the initiation of spontaneous labor at term,3 it is informative to examine the effect of pharmacologically induced P withdrawal (Pw) on the unstimulated preterm uterus. A great variety of studies, in both laboratory animals and patients, indicate that pregnancy cannot withstand Pw of a critical degree.3 This is probably so because throughout gestation the uterus is equipped with an endogenous stimulatory mechanism which triggers uterine activity whenever Pw is excessive. The present method of inducing Pw has been studied earlier in the rat, monkey, and sheep,4 with use of synthetic steroids: androstano[2,3-&Jisoxazols. These anti-Ps (A-Ps) predictably terminate mid823
824
Csapo and Resch
Table I. The effect of isoxazol of labor and fetal weight
without
and with P treatment
Animal’s treatment
No. 12 26 9
Vehicle Isoxazol Isoxazol plus
I,iUU size only
on the time ot onset
onsut
10.7 c 0.7 9.7 2 0.4 11.1 -+ 0.7
if lab,(hr.)
29.1 + 2.2 q (J f 2.2” 3:s: I It 1. I
P
All values are mean5 ? standard error. Dose of oral isoxazol = 20 mg and that of intramuscular calculated from 0 hour on day 2 I, *Significantly different from the control values at the level of P < 0.00 1.
Table
II. The effect of isoxazol
on uterine
tissue P, E,, and PGF levels Time after
0 hr. (h*)
Treatment Progesterone
P = 5 mg. The oncct ol‘labor is
treatment
12 //I. (hl
24 hr.
48 /I?-.
(8)
(-5)
42 i- 3:
30 + 2t
15 t 2$
27 t 49”
22 + 2$’
Drlirv?? (9)
f’o,l pnrtum 131
(nglg)
Vehicle
14-
If
14 t 49
17 i- I$
I6 i: YQ
382 k 33t
86 -+ 133
Delivered
290
48
144 * 11$
105 t 91
67 + 8 Isoxazol Esrradeol
Delivered
(Pglg)
Vehicle
207
+ 26
232
t
18
202
+ 18
464
t 4481
493
2 603
273 -t 22
Isoxazol Prostagkdin
k 29
2 lO$
F (nglg)
Vehicle
482
10
95 2 6t
50 t
20
68 -t 7
Isoxazol
102 5 91
135 2 13SY
Delivered
98 *
lit
40
rt: 10
AlI values are means ? standard errors. For the nine isoxazole plus P-treated rats at 48 hours: P = 71 = 7 ng/gm. Ez = 275 2 39 pgigm, and PGF = 89 f 4 ng/gm. *Number of control and isoxazol-treated rats assayed for uterine I’, Ez, and PGF ai the specified rimea tDifference
statistically
significant
compared
to pretreatment
fDifference statistically significant compared to pretreatment ODifference statistically significant compared to pretreatment “Difference BDifference
statisticaly statistically
significant significant
compared compared
to control to control
value
at time
0 (p < 0.05).
value at time 0 (p i 0.001). value at time 0 (p < 0.01).
value (p < 0.05). value (p < 0.01).
trimester pregnancy, by blocking P synthesis through the inhibition of 3P-hydroxysteroid dehydrogenase. Sequential radioimmunoassays (RIAs) in the uterine tissues of 10 day pregnant rats showed that a single oral dose of isoxazol, which predictably terminates pregnancy, significantly reduces P and increases estradiol (E2) and prostaglandin F (PGF) levels.” If the premise were verified that in preterm rats a similar regulatory imbalance and parturition are triggered by this same steroidal A-P and indeed if it could be shown that this induced imbalance is similar to that which precedes spontaneous labor at term, a new therapeutic principle could be exposed, since induction with A-P does not demand exogenous oxytocic stimulation. This contention. that it is the isoxazol-induced Pw which provokes
the precocious regulatory imbalance and parturition, and not some unrecognized side effects of A-P, could be substantiated by evidence that concomitant treatment with P prevents the action of isoxazol.
Material and methods During March, 1978, pregnant Sprague-Dawley rats were palpated at the laboratory on day 19 of pregnancy, to exclude animals with low litter size and thus reduce variations in the onset of labor which might be caused by season and litteg size. After this selection 95 animals remained, from which 3’7 were used as controls, 40 were treated orally with 20 mg isoxazol, and 18 were given 5 mg P in addition to isoxazol. Throughout the study, each rat was kept in an individual cage at a
Volume Number
Induction of preterm labor by antiprogesterone
134 7
I
VEHICLE
ISOXAZOL
Labor
t
21 DAIS
OF
PREGNANCY
22
ISOXAZOL
21 DAIS
No
Lb
t
R
20
+ PROGESTERONE
825
22
OF PREGNANCY
P
20 DAYS
OF
21 PREGNANCY
22
Fig. 1. The effect
of isoxazol on uterine tissue P, EP, and PGF levels in preterm rats. At 9 PM on day 1’3 uterine tissue was collected from six animals, and 31 rats subsequently received vehicle, 26 isoxazol (20 mg) and nine isoxazol plus P (20 f 5 mg). At different times after treatment uterine tissue was collected from each group as indicated by symbols. Numbers over symbols describe the numbers of P, EB, and PGF assays in duplicate. Note the nature and timing of the regulatory imbalance preceding isoxazol-induced preterm and normal term labor and the effect of P in preventing the isoxazol action.
constant temperature (22” C), exposed to 14 hours of light and 10 hours of darkness, and fed a diet of Purina chow. The experiment began at 9 PM on day 19 of pregnancy, when the control animals weighed 363 +- 8 grams and the isoxazol-treated rats weighed 350 * 6 grams. As determined at the end of the study, the litter size of the three groups of animals was similar (Table I). The control animals were treated orally with the vehicle (2 ml 1% aqueous gum tragacanth) while the isoxazol and the isoxazol plus P rats received 20 mg isoxazol suspended in the same vehicle. The P was injected intramuscularly (in oil), in a single 5 mg dose, at the same time as the isoxazol. The animals, were observed, then killed after delivery for further workup. Exceptions were six animals, autopsied before treatment at day 19 of pregnancy, and 19 control animals, 14 isoxazol-treated, and nine isoxazol plus P-treated rats killed at different times after treatment, but before parturition, as described in Table II. The remaining 12 control, 26 isoxazoltreated, and nine isoxazol plus P-treated rats were allowed to deliver spontaneously (Tables I and II). With midnight of d,3y 20 taken as time 0, the time of delivery of the first newborn animal in each mother defined the onset of labor. The weights of the fetuses and placentas were measured, to assess the effect of isoxazol on the birth weight of the newborn animals and placentas. In
nine control and nine isoxazol-treated animals uterine tissue was collected at the completion of delivery, while in three control and three isoxazol-treated rats tissue collection was delayed for -3 hours post partum. The uterine tissues were stored at -20” C before measurement of P, EP, and PGF, as described previously.4 These assays were performed to determine whether the isoxazol-induced regulatory imbalance, preceding the onset of preterm labor, (1) is distinct, (2) is similar to that which precedes spontaneous labor at term, and (3) is prevented from occurring by P treatment. The data were analyzed for statistically significant differences using Student’s t test.
Results Table I shows that 12 control animals, allowed to deliver spontaneously, expelled the first fetus at 29.1 L 2.2 hours (mean 2 standard error) after 0 time (e.g., around 5 AM of day 22 of pregnancy). The weight of these term newborn animals was 6.1 t 0.1 grams. In contrast, the 26 isoxazol-treated rats delivered the first fetus at 9.0 f. 0.4 hours (P consequent regulatory imbalanc-e 1” onipdng pretcrnl labor. During and after delivery the previousl\ high b:? and PGF levels decreased precipitate]\ in both rhc controt and isoxaLol-treated rats but the alread! low I’ levels did not. ‘I’hus, the regulatory changes preceding the onset of labor were similar in the control and isoxazol-treated rats, but they occurred significanti) earlier in the isoxazol-treated animals. ‘l’he precocious onset in the isoxazol-treated rats resulted iI1 the birth of living newborn animals with lower than normal lhesr c.orresponded, IIO\~C\CT. \\ith theit weights: shorter gestational ages. (:urrent studies are determining the long-term survival I-ate. weight gain. maturation, and reproductive performance of rhc isoxazoltreated newborn animals. A prediction of the “seesaw” theory’ that the ~ntiogenous stimulatory mechanism the pregnant uterus can be activated and thus labor can be induced before term by mere Pw has apparently been validated b) the present experiments. The contention that this action was due to Pw, and not to some side effect ol’treatment. xas unequivocally established by the demonstration I hat protective treatment Faith P prevented the isosa& induced precocious PM, r11e cotrsequenr regulaCor\ in)balance, and preterm deliver!. 'l‘hris. it appears that the unpredictabilit)of the outcome of preterm itlduction of labor with ox!-to& drugs might be resolved I,> developing neb’ concepts and procedures tar activating the normal pregnant reti.actorv uterus withoul forced oxytocic stimulation. The urgency of turthel- work is reflected by reliable clinical evidence that at 32 to 35 weeks of pregnancy even the combined procedures of rupturing the membranes and oxytocin stimulation have failed to provoke labor during 24 hours in 83% of the cases.” Furthermore, of 1.000 patients, 93% who were 3 days before their expected days of confinement were delivered during the first attempt at oxytocin in-
Volume Number
Induction of preterm labor by antiprogesterone
134 7
duction, while the remaining 7% who were 7 days before their expected days of confinement required repeated inductton. Of course, forced induction by
REFERENCES 1. Danforth, D. N.: Obstetrics and Gynecology, York, 1977, !
