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Studies on the effects of altered timing of parturition in the rat
Prostaglandins
and
Medicine
2:
141-154,
I979
STUDIES ON THE EFFECTS OF ALTERED TIMING OF PARTURITION IN THE RAT Brian H. Vickery Department of Physiology Institute of Biological Sciences Syntex Research 3401 Hillview Avenue Palo Alto, CA 94304 ABSTRACT Manipulation of the length of gestation with two different agents has been compared and contrasted in the rat. Single subcutaneous injection of 1 mg/rat of PGF2a on day 19 of gestation caused premature parturition of at least Premature parturition was 24 hours in 55% of treated rats. associated with perinatal death and cannibalism of delivered pups. Subcutaneous implantation of pellets of naproxen over days 20-23 of gestation prolonged gestation to 24 days and synchronized onset of parturition compared to placebo-treated controls. Post-mature onset of delivery, in contrast, was associated with increased birth weight of pups and a survival rate of 90%. Post-mature pup deaths were restricted to the last pups delivered, i.e. longest The potential utility of naproxen for manipulagestation. tion of time of parturition in domestic animals is suggested. INTRODUCTION Manipulation of the timing of parturition has long been regarded as a useful management tool for production of food producing animals. Thus, a number of studies have been conducted in cattle, sheep and pigs investigating the parturition inducing properties of synthetic corticosteroids, particularly dexamethasone, (l-9). Prostaglandins with their luteolytic/labor inducing properties were also proposed for this purpose (10-19). A major drawback with both these types of agent derived from the fact that they essentially induce a premature parturition and therefore tend to be associated with low birth weight and retained placentae (4-6, 18, 20). Certain non steroidal antiinflammatory agents have been shown to block uterine contraction, premature labor I41
and saline induced abortion in species ranging from rat through primate including human (21-23). Naproxen (d-2(6 methoxy-2-napthyl) propionic acid) has also been shown to inhibit labor (34,35) and, more recently, to prolong gestation in laboratory rodents (36). It occurred to the present author that manipulation of gestation resulting in a slightly post-mature birthtime and therefore somewhat increased birth weight, as previously shown (36), might provide a management tool without some of the previously noted disadvantages of presently used agents. The present report therefore summarizes and compares the effect of prostaglandin F2~1 (PGF2a) and naproxen on gestation and the outcome of parturition in the rat. MATERIALS AND METHODS Mature Sprague Dawley derived female albino rats weighing 180-200g were obtained from a commercial supplier (Simonsens, Gilroy, CA), housed in air conditioned temperature controlled quarters under lo:14 dark:light illumination schedule and allowed ad libitum access to food and water for a minimum of two weeksbefore being placed under experiment. After this acclimatization period they were housed 4:l with males and daily vaginal lavages followed to determine mating (presence of sperm in vaginal lavage = day 1 of pregnancy) at which time they were removed from males and housed five per cage. On day 17 of pregnancy all animals were manually palpated to confirm continuation of pregnancy and housed individually for the remainder of experiment. On day 19 of pregnancy subgroups of these animals were administered a subcutaneous injection of 1 mg/rat of PGF2a as a solution in propylene glycol or propylene glycol as vehicle control. On day 20 of pregnancy the remainder of the pregnant rats were either implanted subcutaneously at the back of the neck with a compressed pellet of naproxen and cholesterol or were sham implanted. The pellet was composed of 69.5% naproxen and 30.5% cholesterol, to a total weight of 23.8 mg of active ingredient. At 9:00 am on day 23 of pregnancy the pellets were enucleated. Implantation and removal of pellets took place using light ether anesthesia. Starting at 7:30 am on day 21 of pregnancy and continuing every eight hours thereafter for the duration of the experiment the rats were observed for signs of parturition. The time of first appearance of pups was recorded, as were subsequent deliveries. In addition, appearance and viability of pups was noted. On day 25 of pregnancy all dams were sacrificed and necropsied to confirm completeness of delivery. Number of sites of placental attachment were checked 142
I
PGF2ci
Vehicle only
Treatment
44 cannibal ised/ 11 dead at observation 18 dead at observation 14 survived 1 dead at observation 10 survived
55
32
55.6
33.3 11.1
21
22 23
11
all survive all survive 46 48
44.4 55.6
0
22 23
Viability Pups
Number of Pups
Percentage of Dams Delivering
Day of Gestation
Table I Effect of Injection of 1 mg per Rat of PGF,_ on Day 19 of Gestation on the Outcome of Pregn&Ey in the Rat
g
*
7.30 15.30 23.30 7.30 15.30 23.30
23
24
( 2) ( 9) (10)
( 0) ( 0) ( 0)
( 0) ( 0) 1 0)
( 5) ( 9) (10)
I :; 1 4)
14 61 5
13 48 10
15 36
No. of Pups Recovered
i.e. 69.5% active vs 30.5%
2/10 7/10 l/10
o/10 o/10 o/10
o/10 o/10 o/10
l/10 4/10 l/10
9',:: l/l0
No. of Dams Delivering (Cumulative)
of 23.8 mg of naproxen
7.30 15.30 23.30
7z30 15.30 23.30
23
22
7-30 15.30 23.30
Time
22
Day of Gestation
Pellet composed cholesterol.
Naproxen*
Placebo
Treatment
II
Effect of Naproxen Administered Via Subcutaneous Pellet on Days 20-23 of Gestation on the Outcome of Pregnancy in the Rat
Table
%
100 95 0
100 100
100
100 100
Live Pups
against numbers of delivered pups recorded. Discrepant placental sites were recorded as additional cannibalized pups. Mean and standard error of gestational length was calculated on the basis of first observation time when pups were noted for each dam. Prolongation of shortening of gestation was assessed by comparison to the concurrent controls. The gestational data were analyzed using Jonheere's test for nonparametric ordered alternatives (38). In addition a X2 exact contingency test was applied to the data to test for synchrony of onset of parturition. RESULTS Long standing records on the course of gestation in pregnant female rats derived frem the presently utilized colony has established that parturition occurs sometime over the period 22 to 23 days of gestation (21-22 days from day of detection of mating). In contrast to reports about other authors' colonies (35), this appears to be relatively independent of litter size. Administration of vehicle to pregnant female rats on day 19 of gestation had no effect on either the timing of parturition as noted above or on perinatal survival of pups (Table 1). 1 mg of PGF2a, administered subcutaneously on day 19 induced premature parturition in 55.6% of treated dams. Accelerating the onset of parturition by 24 hours resulted in complete mortality of pups, a high proportion (80%) of which were partially to completely cannibalized. Of the 32 pups delivered on day 22 of gestation, 56% were dead at observation. As there is no way to determine whether these pups would have been delivered on day 22 or 23 in the absence of PGFZa administration, the percentage of premature births is unknown. Pups born on day 23 or gestation had a better than 90% survival rate. The pattern of delivery and survivability of pups from pregnant female rats treated with placebo (cholesterol only) pellets was normal and comparable to the previous experiment (Table 2, Fig. 1). Presence of a subcutaneous pellet of naproxen, during days 23-23 of gestation, significantly (~~0.01) retarded onset of delivery, all dams delivering on day 24 of gestation. First pups were observed 22.5 hours after pellet removal. Overall survivability of these postmature pups, which were visually heavier, was good (90%). Pup deaths which did occur were confined to the last ones delivered. Analysis of the grouping of onset of parturition revealed a significant synchronization compared to controls (p
00
20
40
60
80
40
60
80
100
-
12 N 22
IMPLANTED DAY 20
OF PELLET
IMPLANTATION
OF NAPROXEN
ON DAY
20
12 M
PELLET
w
DAY
23
12 N
REMOVED
12 M
i-l
OF GESTATION
1
12 N 24
N = NOON M = MIDflIGHT
AND PELLET ENUCLEATION ON DAY 23 OF PREGNANCY ON GESTATIONAL LENGTH AND PARTURITION IN THE RAT
EFFECT
FIGURE 1
72 M
NAPROXEN
CONTROL
6.6
? z
6.0
6.2
6.4
6.8
c7 iz 3
2
7.0
3 E 2 z
7.2
7.4
7.6
I
DAY OF GESTATION
* 22
FIGURE
2
/
2h
(28)
/E /’
OF GESTATION
OF PUP WEIGHT
1
TOOK PLACE
(
MEAN + SE
MEAN + SE
1
WITH LENGTH
ON WHICH PARTURITION
2;
CORRELATION
Birthweight
Dams
No. of pups for time point
treated
of pups
Dams
of pups
from Naproxen
Birthweight
from Control
dence of difficulty
or prolongation
of labor was observed.
Data produced in a second experiment where pup weight at first observation was recorded is shown in Fig. 2. It can be seen that the visual observation of increased birth weight of pups born on day 24 of gestation was confirmed. Mean weight of pups born on day 24 was significantly (~~0.01) greater than that of pups from control dams. The continued increase in weight of pups in utero suggests a true prolongation of gestation rather than merely a postponement of labor. DISCUSSION The present experiments have investigated the perinatal effect of manipulating length of gestation in the rat with two very different agents. PGFza was demonstrated to induce a premature parturition in more than half the treated dams, following a single subcutaneous injection of 1 mg per rat on day 19 of gestation. PGF2a has been shown to be both a potent myometrial stimulant (38) and a potent luteolytic agent in the rat (39). Either of these activities could theoretically account for induction of premature parturition, however, the approximately 48 hour latent period between injection and first deliveries would suggest the mechanism to be indirect (luteolysis) rather than direct This is in keeping with the results of (uterotonic). Strauss et al (40) who demonstrated a premature parturition, preceeded by an early fall in circulating levels of progesterone, in rats treated twice daily with 5OOpg of PGF2cton days 19 and 20 of pregnancy. Similar data have been generated by infusion of PGFza (41). The extreme adverse effects on pup survival observed in the present experiment after treatment of dams with PGF,cu. is at variance with the data reported by Strauss et al (40). The discrepancy may be due to differences in treatment schedule, strain sensitivity, or to degree of prematurity. Naproxen has been shown in the present experiments to significantly postpone the onset of parturition in the rat. In contrast to previous reports dealing with the effect of naproxen and other non-steroidal antiinflammatory agents such as aspirin and indomethacin (25,27,40,42), labor did not appear to be prolonged and fetal survival was good. This probably was the result of low level continuous administration of drug, which was abruptly terminated following pellet enucleation, as previously suggested (36). The rapid decay of biological levels, below threshold for interference with myometrial contractility, is reflected by the synchrony of-onset of parturition in the present experiment. 148
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Jochle, W. Corticosteroid Induced Parturition in Mechanism of Action and Economic Domestic Animals: Folia Vet. Lat. 1: 229-259 (1971). Importance.
2)
Liggins, G. C. Premature Parturition after Infusion of Corticotrophins or Cortisol into Foetal Lambs. (1968). J. Endocrinol. -42: 323-329.
3)
Liggins, G. C. Premature Delivery of Foetal Lambs InJ. Endocrinol. 45: 515fused with Glucocorticoids. 523. (1969).
4)
Adams, W. M. The Elective Induction of Labor and Parturition in Cattle. J. Amer. Vet. Med. Ass. 154: 261265. (1969).
5)
Adams, W. M. And Wagner, W. C. The Role of Corticoids in Parturition. Biol. Reprod. 3: 223-228. (1970).
6)
Garverick, H. A., Day, B. N., Mather, E. D., Gomez, L. and Thompson, G. B. Use of Estrogen with Dexamethasone for Inducing Parturition in Beef Cattle. J. Anim. Sci. (1974). -38: 584-590.
7)
Bose, M. J. The Control of Parturition in the Ewe. Proc. Sot. Study Fertil. -27: 491. (1971).
8)
qosc, M. J. The Induction and Synchronization of Lambing with the Aid of Dexamethasone. J. Reprod. Fertil. -28: 347-357. (1972).
9)
Coggins, E. G. and First, N. L. Fetai Decapitation, Dexamethasone and Methalibure Treatment of Pregnant Gilts. J. Anim. Sci. -41: 347 (Abstract) (1975).
10)
Inskeep, E. K. Potential Uses of Prostaglandins in Control of Reproductive Cycles of Domestic Animals. J. Anim. Sci. -36: 1149-1150. (1973).
11)
Liggins, G. C., Grieves, S. A., Kendall, J. Z., and Knox, B. S. The Physiological Roles of Progesterone Estradiol 178 and Prostaglandin F,, in the Control of Ovine Parturition. J. Reprod. Fert. Suppl. -16: 85-103. (1968).
12)
Liggins, G. C. Endocrine Factors in Labour. Endocrinol. -20: 119-139. (1973)
131
Diehl, J. R., Godke, R. A., Killian, D. B. and Day, 150
Mem. Sot.
As the previously noted experiments (25,27,40,42) utilized episodic (once or twice daily) administration, interference with the actual course of labor could be due to either administration during presymptomatic phases of labor or a longer decay of biological levels because of the higher dosage administered. Certain non-steroidal antiinflammatory agents including naproxen inhibit prostaglandin biosynthesis (43,441. Thus naproxen has been shown to suppress PG synthesis by the pregnant rat uterus when administered in vivo (45). The ability of PGF2a to cause luteolysis, in specKsuch as the rat, as well as to stimulate uterine contractility in vitro, has led to some debate on the relative involvement ofther inhibition of luteolysis or uterine contractility in the mechanism of postponement of labor (40,42,46,47). Both mechanisms may play a part in the rat, depending on time of initiation of dosing (36). In the present experiment, the low dosage employed relative to that required to inhibit myometrial contractility in vivo in the rat (26) would suggest that prolongation oflzl function occurred. The increased birth weight of pups from the naproxen treated dams would also favor a true prolongation of gestation rather than inhibition of the mechanics of parturition. The studies reported here point up some of the potential dangers and also benefits of manipulating time of parturition. The utility of naproxen for controlling time of parturition in large animal species has yet to be proven. However, if the synchronization of time of onset of parturition and the increased birth weight obtained in the rat were to be demonstrated in larger animals, naproxen could be a substantial addition to the management armamentarium.
I thank W. Briones for technical assistance with the experiments. 149
B. N., Induction of Parturition in Swine with Prostaglandin F2a. J. Anim. Sci. -38: 1229-1234. (1974). 14)
Ash, R. W. and Heap, R. B. The Induction and Synchronization of Parturition in Sows Treated with ICI 79,939, an Analogue of Prostaglandin F2~. J. Agri. Sci. -81: 365-368.
15)
Einarsson S., Gustafsson B., and Larsson, K. Prostaglandin Induced Parturition in Swine with Some Aspects on Prevention of the MMA (Metritis, Mastitis, Agalactia) syndrome. Nord. Vet. Med. -27: 429-436. (1975).
16)
Henricks, D. N. and Handlin, D. L. Induction of Parturition in the Sow with Prostaglandin F2a. Theriogenology L: 7-14. (1974).
17)
Killian, D. B. and Day, B. N. Controlled Farrowing with Prostaglandin Fnct. J. Anim. Sci. -39: 214. (1974).
18)
Downey, B. R., Conlon, P. D. and Baker, R. D. Controlled Farrowing Program Using Prostaglandins. J. Anim. Sci. -41: 349 (Abstract). (1975).
19)
Gustafsson B., Einarsson, S., Larsson, K., and Edqvist, L. E., Sequential Changes of Estrogens and Progesterone at Prostaglandin-induced parturition in the sow. Am. J. Vet. Res. -37: 1017-1020. (1976).
20)
Lauderdale, J. W. Effects of PGF2a on Pregnancy and Estrous Cycle of Cattle. J. Anim. Sci. -35: 264 (Abstract). (1972).
21)
Vickery, B. H., and Garay, G. L., Mechanism of Action of Non Steroidal Antiinflammatory Agents Inhibiting Uterine Smooth Muscle Motility. Federation Proceedings -33: 575 (Abstract). (1974).
22)
Vickery, B. H., Garay, G. L. And Briones, W., Inhibition of Prostaglandin and Oxytocin Action on the Uterus by Naproxen. Abstracts of the 58th Meeting of the Endocrine Sot. P. 76.
23)
Csapo, A. I. and Csapo, E. E., The "Prostaglandin Step", a Bottleneck in the Activation of the Uterus. Life Sciences -14: 719-724. (1974).
24)
Vickery, B., Garay, G. and Briones, W., Increased Gestational Length due to Naproxen in the Rat in the Absence of Prolonged Parturition. Program of the Ninth Annual Meeting of the Society for the Study of Reproduction, University of Pennsylvania, Philadelphia 151
(Abstract).
(1976).
25)
Aiken, J. W., Aspirin and Indomethacin Prolong Parturition in Rats: Evidence that Prostaglandins Contribute to Expulsion of Fetus. Nature 240: 21-25. (1972).
26)
Vickery, B. H., Effects of Naproxen (in preparation). tility -In Vivo.
27)
Chester, R., Dukes, M., Slater, S. R. and Walpole, A. L Delay of Parturition in the Rat by Antiinflammatoky Agents Which Inhibit the Biosynthesis of Prostaglandins. Nature, Lond. 240: 37-38. (1972).
28)
NOW, M. J., Cook, M. J. and Manaugh, L. Indomethacin Block of Normal Onset of Parturition in Primates. Am. J. Obstet. Gynec. 118: 412-416.
29)
Lewis, R. B. and Schulman, J. D., Aspirin and Labour. Lancet -ii: 1159. (1973).
30)
Zuckerman, H., Reiss, U. and Rubinstein, I., Inhibition of Human Premature Labor by Indomethacin. Obstet & Gynec. -44: 787-792. (1974).
31)
Wizvist N., Lundstrom, V., and Green, K., Premature Labor and Indomethacin. Prostaglandins -10: 515-526.
32)
Waltman R., Tricomi, V., and Palav, A. B., Midtrimester Hypertonic Saline-Induced Abortion: Effect of Indomethacin on Induction/Abortion Time. Am. J. Obstet. Gynec., 114: 829-831. (1972).
33)
Waltman, R., Tricomi, V., and Palav, A. B., Aspirin and Indomethacin: Effect on Instillation/Abortion Time of Mid Trimester Hypertonic Saline-Induced Abortion. Prostaglandins -3: 47-58.
34)
Csapo, A. I., Csapo, E. F., Fay, E., Henzl, M. R. and Salau, G., The Delay of Spontaneous Labor by Naproxen in the Rat Model. Prostaglandins -3: 827-837. (1973).
35)
Csapo, A. I., Csapo, E. F., Fay, E., Henzl, M. R. and Salau, G. The Role of Estradiol 178 in the Activation of the Uterus During Premature Labor and the Effect of Naproxen and Inhibitor of Prostaglandin Synthesis. (1973). Prostaglandins 3: 839-846.
36)
Vickery, B. H., Prolongation of Gestation in the Rat and Hamster by Naproxen. (in preparation). 152
on Uterine Contrac-
37)
Hollander and Wolfe. Non Parametric Statistical Methods. John Wiley and Sons. (1973).
38)
Kirton, K. T., Wyndarden, L. J. and Bergstrom, K. K. Prostaglandins and Myometrial Contractility. Adv. Biosci., 9: 651-655. (1973).
39)
Pharriss, B. B., Tillson, S. A. and Erickson, R. R. Prostaglandins in Luteal Function. Rec. Progr. Horm. Res. 28: 51-89. (1972).
40)
Strauss, J. F. III, Sokoloski, J., Caploe, P., Duffy, P ., Mintz, G. and Stambaugh, R. L. On the Role of Prostaglandins in Parturition in the Rat. Endocrinology 1040-1043. (1975). -96:
41)
Buckle, J. W. and Nathanielsz, P. W. The Effect of Low Doses of F,a Infused into the Aorta of Unrestrained Pregnant Rats: Observations on Induction of Parturition and Effect on Plasma Progesterone ConProstaglandins 4: 443-457. centration. (1973).
42)
Fuchs, A. R., Smitasiri, Y. and Chantharaksri, J. The Effect of Indomethacin on Uterine Contractility and Luteal Regression in Pregnant Rats at Term. J. Reprod. Fert. -48: 331-340. (1976).
43)
Vane, J. R. Inhibition of Prostaglandin Synthesis as as a Mechanism of Action for Aspirin-Like Drugs. Nature New Biol. 231: 232-235. (1971).
44)
Tomlinson, R. V., Ringold, H. J., Qureshi, M. C. and Forchielli, E., Relationship Between Inhibitors of Prostaglandin Synthesis and Drug Efficacy: Support for the Current Theory on Mode of Action of AspirinLike Drugs. Biochem. Biophys. Res. Commun. -46: 552559. (1972).
45)
Williams, K. I. and Vane, J. R., Inhibition of Uterine Motility: The Possible Role of the Prostaglandin and Aspirin-Like Drugs. Pharmac. Therap. B., J_ 89-113. (1975).
46)
Csapo, A. I. and Henzl, M. R., The Mechanism of Naproxen Action in Parturient Rats. Prostaglandins -14: 799-802. (1977).
47)
Kordts E., and Jochle, W. Induced Parturition in Dairy Cattle: Comparison of a Corticoid (Flumethasone) and a Prostaglandin (PGEzcx) in Different Age Groups. Theriogenology -3: 171-178.
153
and
Medicine
2:
141-154,
I979
STUDIES ON THE EFFECTS OF ALTERED TIMING OF PARTURITION IN THE RAT Brian H. Vickery Department of Physiology Institute of Biological Sciences Syntex Research 3401 Hillview Avenue Palo Alto, CA 94304 ABSTRACT Manipulation of the length of gestation with two different agents has been compared and contrasted in the rat. Single subcutaneous injection of 1 mg/rat of PGF2a on day 19 of gestation caused premature parturition of at least Premature parturition was 24 hours in 55% of treated rats. associated with perinatal death and cannibalism of delivered pups. Subcutaneous implantation of pellets of naproxen over days 20-23 of gestation prolonged gestation to 24 days and synchronized onset of parturition compared to placebo-treated controls. Post-mature onset of delivery, in contrast, was associated with increased birth weight of pups and a survival rate of 90%. Post-mature pup deaths were restricted to the last pups delivered, i.e. longest The potential utility of naproxen for manipulagestation. tion of time of parturition in domestic animals is suggested. INTRODUCTION Manipulation of the timing of parturition has long been regarded as a useful management tool for production of food producing animals. Thus, a number of studies have been conducted in cattle, sheep and pigs investigating the parturition inducing properties of synthetic corticosteroids, particularly dexamethasone, (l-9). Prostaglandins with their luteolytic/labor inducing properties were also proposed for this purpose (10-19). A major drawback with both these types of agent derived from the fact that they essentially induce a premature parturition and therefore tend to be associated with low birth weight and retained placentae (4-6, 18, 20). Certain non steroidal antiinflammatory agents have been shown to block uterine contraction, premature labor I41
and saline induced abortion in species ranging from rat through primate including human (21-23). Naproxen (d-2(6 methoxy-2-napthyl) propionic acid) has also been shown to inhibit labor (34,35) and, more recently, to prolong gestation in laboratory rodents (36). It occurred to the present author that manipulation of gestation resulting in a slightly post-mature birthtime and therefore somewhat increased birth weight, as previously shown (36), might provide a management tool without some of the previously noted disadvantages of presently used agents. The present report therefore summarizes and compares the effect of prostaglandin F2~1 (PGF2a) and naproxen on gestation and the outcome of parturition in the rat. MATERIALS AND METHODS Mature Sprague Dawley derived female albino rats weighing 180-200g were obtained from a commercial supplier (Simonsens, Gilroy, CA), housed in air conditioned temperature controlled quarters under lo:14 dark:light illumination schedule and allowed ad libitum access to food and water for a minimum of two weeksbefore being placed under experiment. After this acclimatization period they were housed 4:l with males and daily vaginal lavages followed to determine mating (presence of sperm in vaginal lavage = day 1 of pregnancy) at which time they were removed from males and housed five per cage. On day 17 of pregnancy all animals were manually palpated to confirm continuation of pregnancy and housed individually for the remainder of experiment. On day 19 of pregnancy subgroups of these animals were administered a subcutaneous injection of 1 mg/rat of PGF2a as a solution in propylene glycol or propylene glycol as vehicle control. On day 20 of pregnancy the remainder of the pregnant rats were either implanted subcutaneously at the back of the neck with a compressed pellet of naproxen and cholesterol or were sham implanted. The pellet was composed of 69.5% naproxen and 30.5% cholesterol, to a total weight of 23.8 mg of active ingredient. At 9:00 am on day 23 of pregnancy the pellets were enucleated. Implantation and removal of pellets took place using light ether anesthesia. Starting at 7:30 am on day 21 of pregnancy and continuing every eight hours thereafter for the duration of the experiment the rats were observed for signs of parturition. The time of first appearance of pups was recorded, as were subsequent deliveries. In addition, appearance and viability of pups was noted. On day 25 of pregnancy all dams were sacrificed and necropsied to confirm completeness of delivery. Number of sites of placental attachment were checked 142
I
PGF2ci
Vehicle only
Treatment
44 cannibal ised/ 11 dead at observation 18 dead at observation 14 survived 1 dead at observation 10 survived
55
32
55.6
33.3 11.1
21
22 23
11
all survive all survive 46 48
44.4 55.6
0
22 23
Viability Pups
Number of Pups
Percentage of Dams Delivering
Day of Gestation
Table I Effect of Injection of 1 mg per Rat of PGF,_ on Day 19 of Gestation on the Outcome of Pregn&Ey in the Rat
g
*
7.30 15.30 23.30 7.30 15.30 23.30
23
24
( 2) ( 9) (10)
( 0) ( 0) ( 0)
( 0) ( 0) 1 0)
( 5) ( 9) (10)
I :; 1 4)
14 61 5
13 48 10
15 36
No. of Pups Recovered
i.e. 69.5% active vs 30.5%
2/10 7/10 l/10
o/10 o/10 o/10
o/10 o/10 o/10
l/10 4/10 l/10
9',:: l/l0
No. of Dams Delivering (Cumulative)
of 23.8 mg of naproxen
7.30 15.30 23.30
7z30 15.30 23.30
23
22
7-30 15.30 23.30
Time
22
Day of Gestation
Pellet composed cholesterol.
Naproxen*
Placebo
Treatment
II
Effect of Naproxen Administered Via Subcutaneous Pellet on Days 20-23 of Gestation on the Outcome of Pregnancy in the Rat
Table
%
100 95 0
100 100
100
100 100
Live Pups
against numbers of delivered pups recorded. Discrepant placental sites were recorded as additional cannibalized pups. Mean and standard error of gestational length was calculated on the basis of first observation time when pups were noted for each dam. Prolongation of shortening of gestation was assessed by comparison to the concurrent controls. The gestational data were analyzed using Jonheere's test for nonparametric ordered alternatives (38). In addition a X2 exact contingency test was applied to the data to test for synchrony of onset of parturition. RESULTS Long standing records on the course of gestation in pregnant female rats derived frem the presently utilized colony has established that parturition occurs sometime over the period 22 to 23 days of gestation (21-22 days from day of detection of mating). In contrast to reports about other authors' colonies (35), this appears to be relatively independent of litter size. Administration of vehicle to pregnant female rats on day 19 of gestation had no effect on either the timing of parturition as noted above or on perinatal survival of pups (Table 1). 1 mg of PGF2a, administered subcutaneously on day 19 induced premature parturition in 55.6% of treated dams. Accelerating the onset of parturition by 24 hours resulted in complete mortality of pups, a high proportion (80%) of which were partially to completely cannibalized. Of the 32 pups delivered on day 22 of gestation, 56% were dead at observation. As there is no way to determine whether these pups would have been delivered on day 22 or 23 in the absence of PGFZa administration, the percentage of premature births is unknown. Pups born on day 23 or gestation had a better than 90% survival rate. The pattern of delivery and survivability of pups from pregnant female rats treated with placebo (cholesterol only) pellets was normal and comparable to the previous experiment (Table 2, Fig. 1). Presence of a subcutaneous pellet of naproxen, during days 23-23 of gestation, significantly (~~0.01) retarded onset of delivery, all dams delivering on day 24 of gestation. First pups were observed 22.5 hours after pellet removal. Overall survivability of these postmature pups, which were visually heavier, was good (90%). Pup deaths which did occur were confined to the last ones delivered. Analysis of the grouping of onset of parturition revealed a significant synchronization compared to controls (p
00
20
40
60
80
40
60
80
100
-
12 N 22
IMPLANTED DAY 20
OF PELLET
IMPLANTATION
OF NAPROXEN
ON DAY
20
12 M
PELLET
w
DAY
23
12 N
REMOVED
12 M
i-l
OF GESTATION
1
12 N 24
N = NOON M = MIDflIGHT
AND PELLET ENUCLEATION ON DAY 23 OF PREGNANCY ON GESTATIONAL LENGTH AND PARTURITION IN THE RAT
EFFECT
FIGURE 1
72 M
NAPROXEN
CONTROL
6.6
? z
6.0
6.2
6.4
6.8
c7 iz 3
2
7.0
3 E 2 z
7.2
7.4
7.6
I
DAY OF GESTATION
* 22
FIGURE
2
/
2h
(28)
/E /’
OF GESTATION
OF PUP WEIGHT
1
TOOK PLACE
(
MEAN + SE
MEAN + SE
1
WITH LENGTH
ON WHICH PARTURITION
2;
CORRELATION
Birthweight
Dams
No. of pups for time point
treated
of pups
Dams
of pups
from Naproxen
Birthweight
from Control
dence of difficulty
or prolongation
of labor was observed.
Data produced in a second experiment where pup weight at first observation was recorded is shown in Fig. 2. It can be seen that the visual observation of increased birth weight of pups born on day 24 of gestation was confirmed. Mean weight of pups born on day 24 was significantly (~~0.01) greater than that of pups from control dams. The continued increase in weight of pups in utero suggests a true prolongation of gestation rather than merely a postponement of labor. DISCUSSION The present experiments have investigated the perinatal effect of manipulating length of gestation in the rat with two very different agents. PGFza was demonstrated to induce a premature parturition in more than half the treated dams, following a single subcutaneous injection of 1 mg per rat on day 19 of gestation. PGF2a has been shown to be both a potent myometrial stimulant (38) and a potent luteolytic agent in the rat (39). Either of these activities could theoretically account for induction of premature parturition, however, the approximately 48 hour latent period between injection and first deliveries would suggest the mechanism to be indirect (luteolysis) rather than direct This is in keeping with the results of (uterotonic). Strauss et al (40) who demonstrated a premature parturition, preceeded by an early fall in circulating levels of progesterone, in rats treated twice daily with 5OOpg of PGF2cton days 19 and 20 of pregnancy. Similar data have been generated by infusion of PGFza (41). The extreme adverse effects on pup survival observed in the present experiment after treatment of dams with PGF,cu. is at variance with the data reported by Strauss et al (40). The discrepancy may be due to differences in treatment schedule, strain sensitivity, or to degree of prematurity. Naproxen has been shown in the present experiments to significantly postpone the onset of parturition in the rat. In contrast to previous reports dealing with the effect of naproxen and other non-steroidal antiinflammatory agents such as aspirin and indomethacin (25,27,40,42), labor did not appear to be prolonged and fetal survival was good. This probably was the result of low level continuous administration of drug, which was abruptly terminated following pellet enucleation, as previously suggested (36). The rapid decay of biological levels, below threshold for interference with myometrial contractility, is reflected by the synchrony of-onset of parturition in the present experiment. 148
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