Preclinical evaluation of intrauterine progesterone as a contraceptive agent III. Embryology and toxicology

CONTRACEPTION

PRECLINICAL EVALUATION OF INTRAUTERINE PROGESTERONE AS A CONTRACEPTIVE III. EMBRYOLOGY AND TOXICOLOGY

AGENT

R. Hudson,

B.B. Pharriss, S.A. Tillson ALZA Corporation Palo Alto, CA 94304 and

Hazelton

F. Reno Laboratories Incorporated Vienna, VA 22180

ABSTRACT After insemination, a total of 97 female rabbits had surgical insertions of intrauterine progesterone-delivering systems placebo (nonhormonal) systems, or sham surgery, (10 ug/day), on either day 6 or day 9 of gestation. Of the 65 rabbits treated on day 6, 30 were allowed to deliver naturally or killed on day 39; the remainder were killed on day 29 or 30 following cesarean section. The 32 rabbits subjected to procedures on day 9 were allowed to deliver naturally. No consistent, progesterone-related findings were evident in any part of the study in maternal animals, fetuses, or neonates. No significant treatment-related differences were observed in number and placement of implantation sites, resorption sites, live vs. dead fetuses, or fetal weight and length, either comparing right (inserted) versus left (untreated) horns, or treated versus sham-operated groups. There was no indication of a teratogenic effect following visceral (Wilson's) examinations or skeletal (alizarin) examinations. A special examination showed that neonates from does with system- or placeboinsertions were comparable to control neonates in position of the heart and major vessels. It was concluded that in this study, intrauterine release of exogenous progesterone during gestation is not detrimental to the developing rabbit embryo or neonate. Accepted

for publication

MAY 1978VOL. 17 NO. 5

April

4, 1978

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INTRODUCTION Progesterone actions several investigators to determine whether when applied ment, gestation.

on ova develcpment have (1,2,3,4). The purpose the hormone also affects shortly after implantation

been reported by of this study was embryo developand throughout

In vitro studies show that progesterone can inhibit cleavage -7 z rabbit ova up to the morula staqe, but that it has no effect on the grcwth o-f blastocysts (1). Furthermore, this inhibition is reversible, in that ova incubated in a progesterone-containing medium will divide normally when subsequently placed in progesterone-free medium.

a

In vivo studies also show that progesterone inhibits egg develop-__ ment by an unidentified mechanism. Chanq (2) reoorted that rabbitsgiven 1 mg progesterone subcutaneously two days prior to and again on the day of insemination had only 9% of the normal number of eggs in the uterus six days post-insemination. This subfertility may be due to the rapid transport of the egg to the uterus rather than a direct effect of progesterone on the egg Per In another studv, se. progesterone (57 ug/day) was administered placed in utero in rabbits for 16 days before insemK capsules ination. Eggs of suChanimals examined subsequently (day 6 after insemination) were smaller than those of animals wearing an empty capsule (5 mm). It was noted, however, that many eggs recovered from the proqesterone-treated uteri may not have been fertilized Chang (4) suggested that progesterone affects the synchrony (3). of the female reproductive system, preventing development of eggs and implantation. Allen and Foote (5) reported that eggs recovered from progesterone-treated does and then placed in synchronized pseudopregnant rabbits showed a reduction in implantation compared with eggs from untreated controls. Those ova from progesterone treated does that did implant, however, had a survival rate at term exceeding that of ova from controls (90% vs 68%). Birth weight of pups averaged the same in both groups and postnatal (78% vs 64%). survival was similar

METHODS This study was divided into two parts (Table I), each involving post-insemination surgical insertion of progesterone-releasing systems or placebo systems (no hormone) into rabbit uteri, where they remained throughout gestation. The control (sham) groups had surgery but received no systems. In both exper imerits, maternal health and fetal/embryonic devel.opment were evaluated in detail. Experiment two was designed to provide more detailed data on cardiovascular development of neonates.

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TABLE DISTRIBUTION

OF ANIdALS

Study Part

II Natural

IN PART I AND PART II OF THE STUDY

Group 1 (Sham)

I Cesarean Section Natural Delivery

(CS) (ND)

Delivery

I

Group 2 (Placebo)

Group 3 (Progesterone)

10 10

10 10

15 10

11

10

11

Experiment I: Sixty-five mature New Zealand does were stimulated to ovulate by administration of pituitary luteinizing hormone and impregnated using artificial insemination techniques similar to those described by Gibson -et al. (6). The day of insemination was designated as day 0 of gestation. The does were arbitrarily placed in one control and two test groups (Table I); approximately half were designated for cesarean delivery, while the other half were allowed to deliver naturally. On day 6 of gestation the does were anesthetized with pentothal soaium and a midline laparotomy was performed using sterile technique. Systems (3 mm x 13 mm) releasing 10 pg/day progesterone were inserted into the uteri of 25 rabbits and nonhormonal systems (also 3 mm x 13 mm) into 20 others through a small incision in the rignt uterine horn, where they were held in place witn 5-O Ethilon'suture. The systems have been previously described (7). A sham operation was performed on 20 control rabbits by briefly inserting a probe into the uterus and closing the incision with 5-O Ethilon' suture. Does were delivered by cesarean section on days 29 or 30 of gestation. Does that did not become pregnant, but that had been scheduled for natural aelivery, were killed on day 39, as were does oelivering naturally, but experiencing total offspring mortality by day 39. After the cesarean sections, the following ooservations were recoroed: number of corpora lutea, number and placement of uterine implantation and resorption sites, number and placement of live and dead fetuses, individual fetal weight and length (crown-rump), and external fetal anatomy. Gross necropsies, with examination of uterine and visceral structures, were performed on all does.

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Following external observations, approximately half the fetuses from each litter were fixed in Bouin's solution and evaluated for visceral alterations by the technique of Wilson et al. (8). A cross-section from the head, thoracic, and abdominalregions was taken and examined for abnormalities under the dissecting microscope. The remainder underwent skeletal examination and evaluation for relative differences in size, location, normal or abnormal bone structure, degree of ossification, and the presence or absence of bone structure. The does designated for natural delivery were to rear their young to weaning.

Experiment II: After 32 New Zealand rabbits were stimulated to ovulate as described previously, they were selected at random to have sham surgical insertions, or insertions of a placebo or 10 ug/day progesterone-releasing system, on day 9 of gestation (Table I). The insertions were done as previously described, except that if the right horn was not gravid the left horn, if gravid, was employed. Neonates were weighed, measured (crown-rump), observed for external irregularities, and killed (Diabutal overdose) at the time of first observation after birth. All were subjected to gross necropsy and to a special examination of the heart and aorta by a technique similar to that described by Monie -et al. (9). Maternal animals were killed within 5 days postpartum or by day 36 of gestation. Gross necropsies were performed and close examination made of the uterus and ovaries. Statistical procedures for data evaluation were taken from Morrison (10). RESULTS Experiment I: in 'Table II.

The

disposition

of maternal

rabbits

is presented

TABLE II

PLACEBO

DISPOSITION OF DOES RECEIVING SHAM SURGERY OR OR 10 ug/DAY PROGESTERONE SYSTEMS ON DAY 6 OF GESTATION Group 1 (Sham)

Group 2 (Placebo)

Group 3 (Progesterone)

20

20

25

Animals

Inseminated

Animals

Pregnant

(CS)*

5

5

9

Animals

Pregnant

(ND)**

3

4

3

Maternal

Deaths

(CS)

1

2

1

Maternal

Deaths

(ND)

2

0

0

* Cesarean Section ** Natural Delivery

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'The 6 deaths were unrelated to treatment regimen. Individual uterine and ovarian data for pregnant animals scheduled for cesarean delivery showed slight reductions in the ratio of implantation sites to corpora lutea in the right (surgically treated) horn compared to the left (untreated) for sham- and placebo-treated groups; the reverse was true, however, for the A summary is presented in Table progesterone-treated group. III of the observations of uteri and litters of all does delivering by cesarean section. TABLE III SUMNARY OF MEAN UTERINE AND LITTER OBSERVATIONS FOR DOES DELIVERING BY CESAREAN SECTION Group 1 (Sham) Pregnant

Does

Implantation

5

Group 3 (Progesterone)

4*

9

6.6

6.3

5.8

0.2 0.2

0.3 0.5

0.4 0.4

Live Fetuses-Right -Left

2.6 3.6

3.3 2.0

3.1 1.9

Dead Fetuses-Right -Left

0 0

0.3 0

0.1 0.1

Resorption

Sites

Group 2 (Placebo)

Sites-Right -Left

Pup Weight

(gm)-Right -Left

46.2 45.1

47.2 46.9

43.0 42.4

Pup Length

(cm)-Right -Left

9.3 9.6

9.5 9.6

9.3 9.6

* One pregnant

animal died on Day 20 and is not included.

No treatment-related meaningful differences were observed among any parameters shown above. The slight reduction in mean pup weights in the progesterone-treated group is due to one litter of 11 pups (largest in this group). Excluding values for that group renders the group mean comparable with that of the other two. At cesarean section all pups appeared grossly normal except for 2 aead pups from a single doe in Group 3 (progesteroneOne of the 2 showed an inward rotation of the left treated). front leg and lividity in the lower l/2 of the body: the other had dark red hindlimbs, both rotated slightly inward. Neither abnormality was considered treatment related.

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Morphology of the cardiovascular system and other viscera examined by Wilson's technique showed no treatment-related malformations; development was within normal limits in all animals. Skeletal examinations of the fetuses from control and test litters also showed no treatment-related abnormalities. Furthermore, no trends toward lesser or greater development were observed in the progesterone-treated groups compared to the control groups. Delivery and neonatal survival data for does delivering spring naturally are summarized in Table IV.

off-

TABLE IV SURVIVAL DATA FOR OFFSPRING DELIVERED NATURALLY Group 1 (Sham)

Group 2 (Placebo)

Group 3 (Progesterone)

Average Days to Delivery

32.7

32.3

33.0

Total Number of Pups

10

20

14

Live Birth Index

80%

95%

79%

0% 0% 0%

11% 0% 0%

64% 50% 50%

Survival Index Day 7 Day 10 Day 49

Following natural delivery all pups appeared grossly normal, out most were found dead within 4 days postpartum; near complete or partial cannibalization of a majority was evident. 'This occurred in all groups, including the sham-treated group. Table V presents the disposition Experiment II: Zealand does treated on day 9 of gestation.

of the 32 New

TABLE V DISPOSITION OF DOES RECEIVING SHAM SURGERY OR PLACEBO OR 10 ug/DAY PROGESTERONE SYSTEMS ON DAY 9 OF GESTATION Group 1 (Snam) Animals Inseminated

Group 2 Group 3 (Placebo) (Progesterone)

11

10

11

Animals Delivering Naturally

8

2

3

Animals tiot Delivering Naturally

3

6

2

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In group 2, pups of one doe were delivered by cesarean section. The two deaths that occurred were unrelated to treatment regimen; one doe died during a surgical procedure and another during abortion. The remaining does not delivering normally aborted. A summary of the condition of fetuses is shown in Taole VI. Maternal appearance, behavior, survival, weight gains, and necropsy findings of the system-inserted groups were comparable to those of controls. Two placebo-treated animals died of Abortions in 3 sham-treated, 6 causes unrelated to treatment. placebo-treated, and 2 progesterone-treated does were considered a result of surgical stress. TABLE VI A Sili%%AHYOF FETAL PARAMETERS,

PART II OF EXPERIMENT

Mean Fetal Mean Fetal Mean Fetuses NO. of height (gm) Length (cm) ResorpPer Litter Live tion Litters Total Live Dead Live Dead Dead Sham

8

6.4

3.9

2.5

56.6

56.0

10.1

10.4

0.9

Placebo

2

6.5

5.0

1.5

57.3

44.8

10.6

9.6

0.5

Progesterone

9

3.4* 2.0

1.4

63.3

51.9

10.4

11.3

* l

Significantly

lower than control

1.3**

at p< 0.05.

* Elevated due to 7 resorptions found in one litter. litter is excluded from the mean, the value oecomes

If this 0.5.

The numoer of neonates, stillbirths, weights, lengths, and external appearance of progesterone and placebo groups were comparaole to those of the sham-treated group. Rotation of limos was noted in 2 fetuses of one litter of the sham treated One group and 2 fetuses of one litter of the placebo group. fetus of the progesterone group had malformed paws and a downward flexure of the forepaws at delivery. All 5 malformed The heart and major vessels of fetuses were dead at delivery. the inserted and sham-treated groups yielded comparable observations. Apparent malposition of the major vessels was noted in one fetus of a sham-treated animal and one fetus of a placebotreated animal. All fetuses of does that received the progesterone system appeared normal. DISCUSSION In Experiment I, the slight reductions in ratio of implantation sites to corpora lutea in the rignt (surgically-treated) horn to the left (untreated) horn for both the sham and the placebo-

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treated group have no physiological significance. No significant treatment-related differences were observed in number and placement of implantation sites, resorption sites, live vs. dead fetuses, or fetal weight and length, either comparing right (inserted) versus left (untreated) horns, or treated versus sham-operated groups. There was no indication of a teratogenic effect following visceral (Wilson's) examinations or skeletal (alizarin) examinations. No greater or lesser development of any structures characterized control versus treated groups. During gestation rabbits delivering full-term litters showed no effects of devices upon appearance, behavior, and body weight. Following delivery, all pups in each group appeared grossly normal: no anomalies were visible. In Experiment II, no consistent progesterone-related findings were evident in either maternal animals or neonates. The special heart examination showed that neonates from does with system or placebo insertions were comparable to control neonates in position of the heart and major vessels. Apparent malposition of the major vessels occurred in one neonate of a sham-treated rabbit and in one of a placebo-treated rabbit. All neonates of progesterone-treated does appeared normal. The rate of limb flexure in pups for the sham, placebo, and progesterone treatment groups was, respectively, 2 of 86, 2 of 55, and 3 of 96; statistically there are no differences between the groups. In the rabbit, limb flexure is considered a common minor anomaly. It appears to be due to restricted movement within the uterus and with subsequent excercise would disappear (11). The foregoing teratology data indicate that intrauterine progesterone, released at 10 ug/day in the uterine lumen, appears to have no embryotoxic or teratogenic effects in the pregnant rabbit nor is it detrimental to the general well-being of the developing embryo or neonate.

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REFERENCES 1.

Daniel, J.C. and Levy, J. Action of progesterone as a cleavage inhibitor of rabbit ova -in vitro. ,J Reprod Fertil 7: 323-329 (1964)

2.

Chang, M.C. Effects of progesterone and related compounds on fertilization, transportation, and development of rabbit Endocrinology 81: 1251-1260 (1967) eggs.

3.

Seshadri, A., Gibor, Y., and Scommegna, A. Antifertility Am J effects of intrauterine progesterone in the rabbit. Obstet Gynecol 109: 536-541 (1971)

4.

Chang, M.C. Fertilization, transportation and degeneration of eggs in pseudopregnant or progesterone-treated Endocrinology 84: 356-361 (1969) rabbits.

5.

The effect of progesterone Allen, M.C. and Foote, R.H. on the early development of the rabbit embryo. Fertil Steril 24: 220-226 (1973)

6.

Gibson, J.P., Staples, R.E., and Newberne of the rabbit in teratogenicity studies. Pharmacol 9: 398-408 (1966)

7.

Hudson, R., Hemphill, P., and Tillson, S.A. Preclinical evaluation of intrauterine progesterone as a contraceptive agent. I. Local contraceptive effects and their reversal. Contraception 17:465-474 (1978)

8.

Wilson, J.G. and Warkany, J. Teratology: Principles Techniques. University of Chicago Press, 1965

9.

Monie, I.W., Lho, K., and Morgan, J. Supplement 1965, p. 165-167 Teratology Workshop Manual.

to

10.

Morrison, D. Multivariate Hill, New York, 1967

McGraw

11.

Plamer, A.K. In Drugs and Fetal Development (M.A. Klingberg, Editors) Plenum Press, New A. Abramovici, and J. Chemke, York, 1972, p. 45-60

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statistical

J. W. Use Toxic Appl

methods.

and

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