Azathioprine (Imuran) and pregnancy

FETUS AND NEWBORN

Azathioprine JENS JOHN

G.

ROSENKRANTZ,

H.

SHEILA

M.D.

GITHENS, M.

DON.4LD Denver.

(Imuran) and pregnancy

COX, L.

M.D. B.A.

KELLUM,

M.D.

Colorado

The results of treatment of pregnant Swiss-Webster mice with various doses of azathioprine are described. Skeletal anomalies, cleft palate, and apparent decrease in thymic sire were noted in the fetus when the drug was administered during the embryonic period of development. Equally significant, hydrops fetalis, anemia, and severe hematopoietic depression in the absence of significant maternal hematopoietic depression were observed after administration of the drug in certain phases of fetal development. Fertility of male and female animals was not affected by the dose used, nor was the estrus cycle changed. The possible effects of this drug on the embryo and fetus should be considered whenever it is used for immunosuppression among human females of childbearing age.

IMMUNOSUPPRESSIVE drugs have been used with increasing frequency in patients undergoing homotransplantation or with autoimmune diseases. The purine antagonist, azathioprine, Imuran or 6- (methylr-ni tro5-imidazolyl) -thiopurine, has proved a particularly effective agent under these conditions. Since this drug is being used in humans of childbearing age, it is important to know about. its effects on fertility and pregnancy.

Unlike mercaptopurine,ly ?, 3 a closely related analogue, azathioprine has been found to have no teratogenic activity in the rat.4 However, the present study, previously reported in part,5 has demonstrated a profound effect of the drug on pregnancy in the Swiss-Webster mouse. Because of the implications of these findings on the management of patients receiving azathioprine, they are reported here. Material

From the Departments Pediatrics, University Medical Center.

of Surgery of Colorado

methods

studies. animals. Ten male Swiss-Webster mice, 47 to 57 days old, were given 20 mg. per kilogram of azathioprine intraperitoneally, dissolved in dilute NaOH (pH adjusted between 7.8 and 8.6). The drug was administered five times a week for a total of 19 injections.

and

Supported in part by Grant T-138E of the American Cancer Society and part by United States Public Health Service Grants HD-01100 and HE-07741. The azathioprine Burroughs-Wellcome

Fertility

and

Male

in

was supplied by and Company. 387

388

Rosenkrantz

et

February 1, 1967 Am. J. Obst. & Gynec.

al.

Five animals were then bred with virgin females, 13 to 15 weeks old; the other 5 with females which had previously been proved fertile. Injections of azathioprine were continued until a mating plug was found in the female. The males received a total of 21 to 26 injections. Pregnancies resulting from this mating were allowed to continue undisturbed. Four of these males were treated with azathioprine for a total of 30 to 40 days and were bred a second time with other females and the resulting pregnancies were allowed to continue to term. Five litters, resulting from these 14 matings, were raised to maturity, and eight litters were sacrificed at birth and the infants studied for skeletal anomalies by the alizarin red S technique.6 Female animals. The same dose of azathioprine was administered in a similar manner to 18 virgin female mice, 7 to 15 weeks of age. After 19 injections, the females were bred with fertile males and the drug continued until a mating plug was found. The females received a total of 19 to 24 injections. Pregnancy was allowed to continue and, after delivery, two of the litters were raised to maturity, one litter was killed and inspected for skeletal anomalies after staining with alizarin red S, and the remaining litters were not studied further. Teratologic studies. Two or three consecutive daily injections of azathioprine were administered to 10 to 15-week-old pregnant Swiss-Webster mice. Conception was marked by the appearance of a vaginal mating plug and considered to be day 0. The doses of azathioprine administered were 4, 10, 20, and 30 mg. per kilogram; these were given on days O-2, 3-5, 6-8, 9-10, 9-11, 12-14, or 15-17 of gestation. Additional animals received either no injections or intraperitoneal injections of dilute NaOH (pH 8.6) on each day of pregnancy from 0 to 14 and were labeled control and injected control animals, respectively. The products of conception were delivered by hysterectomy at 17 or 18 days of gesta-

tion and evaluated for resorption, fetal death, and runting. Runting was defined as fetal weight less than the mean weight minus two standard deviations for control fetuses of similar age. For purposes of further study, live fetuses were divided by litter into three groups and underwent: (1) staining by the alizarin red S technique and inspection for skeletal anomalies-988 fetuses from 108 litters; (2) preservation in 10 per cent formalin and microdissection to discover external and visceral anomalies-722 fetuses from 73 litters; and (3) hematologic studies, including open aspiration of heart’s blood for cellular morphology and microhematocrit; histologic examination of liver and bone marrow stained with hematoxylin and eosin; and examination of “touch” slides of liver and femoral marrow stained with GiemsaA5 fetuses from 10 litters. In addition, 6 pregnant mice were treated with 30 mg. per kilogram of azathioprine on days 12 to 14 of pregnancy (3 animals) or days 15 to 17 (3 animals), killed on day 18 of pregnancy, and microhematocrit and morphologic examination of peripheral blood and bone marrow carried out. Results

Fertility

studies.

Male animals. The first matings of 9 of the ten males resulted in normal pregnancies; one mating was sterile. Second matings, with 4 males, were fertile. Five of the litters, raised to maturity, showed no external anomalies, although the babies in one litter appeared small. In the 8 other litters, 78 babies were examined for skeletal anomalies. None were found. Female animals. Among the 18 pretreated females, mating plugs were noted during the first estrus in 14 animals (78 per cent). Untreated females had been observed to have mating plugs during the first cycle in 43 of 70 cases (61 per cent). After these 14 matings, normal pregnancy ensued in 9 (64 per cent). Among normal females in this laboratory, 62 to 83 per cent have conceived following mating. Two of

Volnme Number

97 3

Azathioprine

these nine litters were raised to normal maturity without overt anomalies; one litter of 13 animals, stained with alizarin red S, showed no skeletal anomalies. The remaining litters were not studied further. Teratologic studies. Resorptions, deaths, and runting. These data are summarized in Table I. Death of the conceptus was noted in significant numbers when azathioprine was given after the second day of pregnancy; resorption occurred after death early in pregnancy. Runting \Nas observed among many surviving fetuses. Because of hydrops occurring in fetuses whose mothers were treated between days 12 and 14, fetal weights in these animals were not considered indicative of the degree of runting. Alizarin-stained fetuses. Skeletal anomalies among living fetuses were described in Table II and illustrated in Figs. 1, 2, and 3. The Table

and

pregnancy

389

types of skeletal anomalies varied according to the timing of drug administration, and included encephalocele and meningocele among those injected on the third to fifth day of gestation; abnormal tails, rib fusion, hemivertebrae, and vertebral arch anomalies occurred among those injected from the sixth to the eighth day; and oligodactyly, scapulohumeral anomalies, micrognathia, abnormalities of the frontal and parietal bones, and constriction of the fibulas were found in fetuses treated from the ninth to the twelfth day. In addition to these anomalies, variations from normal skeletal anatomy were observed in 76 of the 282 fetuses in the two control groups, including supernumerary ribs (41 cases), additional sternebrae (24 instances) and an extra ossification center in the axis (11 fetuses). These variations also occurred in the treated animals and were not included among the skeletal anoma-

I. Results of pregnancy

~gyq

f!$g;

Control Injected

1

;rie;f

control

o-2

9-

12-14 .___ ‘Difference

between

;;;

1 2 0 3

6 8 6

78 96 81 143

171 20” 34* 65*

3 5*

23* 6 5 17*

1

13*

6 7

95 130 76 89

4* 2 14*

12* 2 13*

4 10 20 30

6 6 9 7

72 69 113 83

9 6 8 5

0 13* 36”

2 4 46* 25*

4 10 20 30

6 8 8 11

73 101 80 111

14’ 5 5 16

3 0 33” 911

2 15* 18” 4*

4 10 20 30

1 2 2 9

11 23 23 103

0

0 3” 0 8*

11

4 10 20 30

11

)

24 11 1 5 201 12

4 10 20 30

9- 10

;:lfr

330 122 65 81 77 92

10

6-8

,,,“d;$?;n, 1

32 9 6 7 6 7

0 4 20 30

3-j

1T;.&$;;;l

experimental

10 10

data

and

control

data

is significant

1

1

3

0

9* IO” 6* 13*

1

0 6 chi

1

1

1

by

9 2 0 9*

square

(P

less than

0.011.

390

Rosenkrantz

February 1, 1967 Am. J. Obst. & Gym.

et al.

Fig. 1. A, Skull skull of normal parietal bone.

of 18 day fetus treated on the third to fifth 18 day fetus; B, demonstrates absence of

lies listed in Table II. There was, however, an increased incidence of these variations in the litters of mothers injected in the 6 to 8 day period with all doses of azathioprine. Microdissected fetuses. The findings among those fetuses subjected to microdissection are summarized in Table III. Cleft palate was frequently seen among live fetuses whose mothers received azathioprine on the ninth and tenth days after conception, but rarely in the group receiving comparable doses in the ninth through the eleventh day. This difference may be explained in part by the high mortality rate after teratogenic doses in the latter group. Dead fetuses were not examined. Decreased thymic size, less than 50 per

day of gestation, compared major portion of occipital

with and

cent of the thymic size in control animals, was apparent among offspring of mothers who received 10 mg. per kilogram or more on the ninth and tenth days, as well as on the ninth through the eleventh day. A large proportion of fetuses whose mothers received 30 mg. per kilogram of azathioprine between the twelfth and the fourteenth day showed marked generalized edema, pallor, and lethargy. Apnea and death occurred after a few ineffectual attempts at respiration. No other abnormalities were noted during microdissection in any groups. Hematologic studies. The marked hydrops of fetuses whose mothers received 30 mg. per kilogram of azathioprine from the

Azathioprine

Fig. 2. Skeleton of sixth to eighth day anom.alies of vertebrae

IFig.

3. Fore-leg

18 day fetus treated of gestation. Shows and rib fusion.

and

shoulder girdle absence of

$on. Note oliqodactyly,

on the multiple

of 18 day

and

pregnancy

391

twelfth through the fourteenth day is demonstrated in Fig. 4. This was present in 18 of the 45 fetuses whose mothers received this dose. Data on fetal weight and hematocrit are given in Table IV. Anemia and an increase in nucleated red cells in the peripheral blood were universal in these animals; the average hematocrit in these fetuses was significantly less than that measured among control and injected control animals. Bone marrow and liver “touches” and sections were studied in 24 of these fetuses and compared with observations in 25 control and 18 injected control animals. There was an increase in hematopoietic activity in the liver, with erythroid hyperplasia and predominance of immature erythroid and granulocytic cells. However, the bone marrow was hypoplastic. This was in contrast to the normal appearance of liver and bone marrow among control and injected control animals. The 19 fetuses of mothers given 30 mg. per kilogram of azathioprine from the fifteenth to the seventeenth day showed no hydrops, and the average hematocrit was normal. Only rare nucleated red cells were observed in the peripheral blood. These animals all showed significant depression of hematopoietic activity in the liver, with

fetus

treated

on ninth

humeral deltoid process, absence

to twelfth day of gestaof scapular spine.

392

Rosenkrantz

February 1, 1967 Am. j. Obst. & Gynec.

et al.

Table II. Skeletal

anomalies

DizE?;;dEly/

~$w;;

/

,+$,

1 e%E~d

1 %ll;s

j Types

Control

-

Injected

o-2

3-5

6-8

9-

10

9-11

12 - 14

III.

Table

20

220

2

0

5

62

0

4

3

30

0

10 20 30

4 3”

40 27 37

8 0

4 10 20 30

3 5 3 8

23 47 23 52

0 1 0 1

4 10 20 30

5 6 3

39 71 37 30

0 4 4 10

Abnormal tails; rib fusion; hemivertebrae ; vertebral arch anomalies

4 10 20 30

3 3 5 4

32 27 47 18

0 14 15 17

Oligodactyly; scapulohumeral anomalies ; micrognathia

4 10 20 30

3 3 5 2

29 33 17 4

0 0 10 4

Oligodactyly; scapulohumeral anomalies; anomalies of frontal and parietal bones; constriction of fibula

30

5

43

0

control

1 Lmd;;

Control Injected

1 ;%&i

-

3-5

6-8

9-10

9-11

14

1 ci$.pwa;;zc

7 4

59 47

4 10 20 30

3 3 3 3

34 33 29 30

4 10 20 30

3 3 3 3

38 28 21 21

4 10 20 30

4, 3 3 3

26 33 32 28

4 10 20 30

3 3 3 3

31 35 33 24

4 10 20

2 4

,3

30 between

1 7;;;

0

control

o-2

*Di*erence

fusion; alies

vertebral

Encephalocele meningocele

anom-

; lumbar

Microdissection

L2i-L;~~

12-

Rib

of anomalies

exPerimental

and

1 No.&

9* 18+ 19”

2”

1 ;;o.$$

2

1

20* 15*

Li; 25

4 dab

j %;;sh

1

15” 18*

46 control

PIUS

injected

control

Ata

is significant

by chi

Sgllare

(p less

35*

than

o.olj.

Volume Number

97 3

Azathioprine

and

pregnancy

393

Table IV

Daysof

pregnancy dose was administered

Daily dose (mg./Kg.)

No. of litters

No. of fetuses

Mean

-

2 2 4 2

25 21 45 19

1.431 1.399 1.397 1.510

Control Injected control 12 - 14 15 - 17 *Significantly

Fetal hematocrit

Fetal weight (grams)

different

0 30 30 from

the

two

control

groups

by

Comment Azathioprine, in the dose and treatment schedule used, did not appear to affect the fertility of either male or female SwissWebster mouse when given prior to mating. Nor did this drug interfere with the estrus cycle, in contrast to the rat treated with 6-

I Mean

0.046 0.140 0.286 0.139

Student’s

t

test

Fig. 4. Hydrops fetalis in 18 day fetuses treated on twelfth

decreased numbers of immature granulocytes and all erythroid cells. The marrow, on the other hand, showed only a slight hypocellularity. The hematocrits and bone marrow of 18 day pregnant adult mice were studied after treatment with the same dose of azathioprine (30 mg. per kilogram), administered in 3 animals from 12 to 14 days of pregnancy and in 3 from 15 to 17 days of gestation. No anemia or other abnormalities were observed in the peripheral blood, although the bone marrow showed slight erythroid hypoplasia in those treated from the fifteenth to the seventeenth day. The marrow of animals given azathioprine on the twelfth to the fourteenth day had apparently recovered and was r,ormal.

(%I

Standard deviation

37.0 37.9 19.8* 38.1 (P

<

Standard I deviation

5.5 9.3 5.2 9.5

No. with hydrops 0 0

18 0

0.01).

to fourteenth

day of gestation.

mercaptopurine.’ Congenital anomalies were not found among the offspring of these males or females treated prior to mating. Azathioprine does exert a significant effect on the embryo of the Swiss-Webster mouse. Death among the offspring, with or without resorption, was common with certain dose schedules. Many live fetuses showed runting. Skeletal anomalies, including cleft palate, were common. Another finding, at certain dose schedules, was the apparent decrease of thymic size. Whether this observation is real and can be quantitated and whether it has any influence on immunologic integrity cannot be determined from this study. Of even more significance to the human is the suppression of hematopoiesis and hydrops observed among the offspring of mice which received large doses of azathioprine late in pregnancy. Such hydrops has been noted previously in teratologic experiments with alkylating agents.7 In the present study, it was invariably associated with anemia, suggesting that the edema may have been due to heart failure, as postulated in humans8 Depressed hematopoiesis was present im-

394

Rosenkrantz

February 1, 1967 Am. J. Obst. & Gym.

et al.

mediately after treatment in these fetuses and seemed at least partially responsible for the severe anemia. Hematopoiesis in the liver and in the bone marrow seemed to respond differently to azathioprine, the liver apparently recovering first from the effects of the drug. Other mechanisms for the development of anemia were not investigated in this study, although the rapid drop in fetal hematocrit would indicate that factors in addition to depressed hematopoiesis were responsible. In any case, these studies suggest that, in the Swiss-Webster mouse, fetal hematopoietic tissue is more susceptible to the effects of azathioprine than adult hematopoietic tis-

sue. If this is true in the human, the treatment of a pregnant woman with azathioprine, even with careful monitoring of her hematopoietic response, could result in an anemic or hydropic fetus. As a general rule, however, the teratogenic effects of a drug vary widely from species to species and even among different strains of the same species. One cannot conclude, therefore, from these or other studies which, if any, anomalies might result from the administration of azathioprine to pregnant humans. Only careful clinical observations will answer questions concerning fertility, fetal death and congenital anomalies in the human treated with azathioprine.

REFERENCES

1. Bragonier, 2. 3. 4. 5.

J. R., Roesky, N., and Carver, M. .T.: Proc. Sot. Exner. Biol. & Med. 116: 685. i964. Thiersch, J. B.: Ann. New York Acad. SC. 60: 220, 1954. Zunin, C., and Borrone, C.: Minerva pediat. 7: 66, 1955. Thiersch, J. B.: J. Reprod. Fertil. 4: 297, 1962. Githens, J. H., Rosenkrantz, J. G., and Tunneck, S. M.: J. Pediat. 66: 959, 1965.

6.

Cumley, R. W., Crow, J, F., and Griffen, A. B.: Stain Technol. 14: 7. 1939. 7. Murphy, L. M., del’Moro, A., and Lacon, C.: Ann. New York Acad. SC. 68: 762, 1957. 8. Allen, F. H., and Diamond, L. K.: New England J. Med. 257: 659, 1957. 4200 East Ninth Denver, Colorado

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