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Experimental production of genital lesions with norethindrone
dume
89
number
6
July 15, 1964
American
Journal
of Obstetrics and Gynecology
GYNECOLOGY Experimental production of genital lesions with norethindrone LAWRENCE ROGER Baltimore,
R. B.
SCOTT,
WHARTON,
JR.,
M.D.
M.D.
Maryland
T H E L A T E Lawson Wilkins and his associates’! 2 in 1958 and again in 1960 reported a series of masculinized infants with apparently normally functioning adrenal glands. The important factor in the production of these anomalies appeared to be the treatment of the pregnant mother with an assortment of hormones. Among 101 cases collected by Wilkins, 34 had received ethister-
one; 35, norethindrone; I, norethynodrel ; 15, testosterone; 2, progesterone; 4, stilbestrol; and 10, no endocrine therapy. In each female infant, the clitoris was enlarged and there were varying degrees of labioscrotal fusion. The infants were normal in all other respects and became normally feminized later with normal menarche, menstruation, and ovulation. All thlls far investigated ha1.e had chromatin-positive buccal smears. In 1962 Jacobson:, treated 385 consecutive pregnant private patients with norethindrone noting a 5.5 per cent incidence of maternal virilization and an 18.3 per cent over-all incidence of fetal virilization. The frequency of these changes rose to 23.7 per cent when norethindrone was started by the twelfth week of pregnancy. Because of the findings of Wilkins and his group our interest was aroused in this subject. The fact that androgens given to pregnant animals can virilize their offspring had been well established in a variety of ani-
From the Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, and the Baltimore, Maryland, and the Department of Obstetrics and Gynecology, Western Reserve Unirlersity School of Medicine, Cleveland, Ohio. This work was supported in part by Research Grant No. AM 0592% of the National Institutes of Health, Nation Institute of Arthritis and Metabolic Diseases. Progesterone in oil was generousl) supplied by Ayerst Laboratories. Norethindrone was generously supplied as No&tin by Parke, Davis B Company.
701
702
Wharton
Table
and
I. Progesterone
Animal 18B 19B 2OB
study Duration pregnancy 167 158 158
Progesterone started (day) 26 24 28 28 ‘8 2-P .__
21B 22B 23B
Table
July 1.5, 1')h.l Am. J. Ohst. B (;ynw.
Scott
II. Norethindrone
Condition at birth
(grams)
152 161 165
Sex
1 Buccal smear chromatin
350 350 400
Living Living Living
0 i
i-+ .
420 400 400
Living Living Living
8 P 8
t -
study i :[I;:
i dr%+;;;;:;ed
___-
--___Birth weight
of
( ii;;)
1 C$X;~--~
1
1 ciE;in
Remarks
--
Animal 32 33 35 31 32 27 35 27 79 2
16B-1 16B-2 18B 19B ?OB-1 “OB-2 21B 22B 23B-1 23B-2
128 131 162 167 133 111 143 96
260 300 550 450 400 300 300 143
128
280
104
mals. Most pertinent to this study was the work of Wells and VanWagenen.4 They treated pregnant Rhesus monkeys with testosterone proprionate in oil beginning on the forty-first to sixty-ninth day of pregnancy and continuing through the ninetyninth day. Two untreated controls were normal, but 9 of 10 treated animals were virilized. The findings consisted of an elongated urogenital sinus and absence of the distal vagina but prostatic tissue and seminal vesicles were found. These changes persisted into adult life, but the animals had a normal menarche and menses. Material
and
methods
With this background information the study was started using the Macaca mulatta initially in the animal colony of the Carnegie Institute of Embryology and later at the Primate Center of the Johns Hopkins University. Personnel5 working with the Carnegie Institute of Embryology animal colony have long been interested in breeding Rhesus monkeys and records are available of many
Stillborn Stillborn Stillborn Living Stillborn Stillborn Living Stillborn Stillborn Macerated
i 8 ; 9 i 0 0
: + + + +
Inguinal testes Intra-abdominal testes Inguinal testes Masculinized Jnguinal testes Masculinized Masculinized Intra-abdominal testes Masculinized Masculinized -.---.
pregnancies which may in a way serve as controls. These animals usually have approximately a 28 day menstrual cycle with ovulation usually occurring about 15 days prior to menstruation. The average duration of a normal gestation is considered to be 160 to 165 days as estimated from ovulation rather than from the preceding menstrual period. The pregnancies usually proceed uneventfully to term, and abortion or stillbirth is unusual. The parturient monkey rapidly eats the placenta making this or,qan usually unavailable for study. The newborn term Rhesus monkey weighs in the nei,ghborhood of 100 grams’ and has well-developed genitals. Usually it is quite easy to distinguish between male and female newborn. Occasionally at birth the testes are undescended, lying in the inguinal area rather than in the scrotum. There are no other genital variants and spontaneous malformations of the reproductive tract have not been observed in this colony. The uterus is normal arcuate in type. Mature female Macaca mulattas, after
Volume Number
89 6
Genital
being received in the colony, were held for a period of observation during which time an attempt was made to correct nutritional deficiencies, improve their general health, and record the character of their menstrual cycles. After this had been accomplished, the animals were mated for usually a period of 4 to 5 days beginning on day 9 or 10 of the menstrual cycle. These animals have a cycle of about 28 days’ duration and frequently do not develop amenorrhea until the second menstrual period following fertilization. For this reason internal genital organs of the animals were palpated following each menstrual period or whenever the cycle was missed. As soon as the pregnancy could be diagnosed by palpation, hormone injections were commenced. In the first part of the study 6 pregnant Rhesus monkeys were treated with progesterone in oil intramuscularly, receiving 50 mg. daily for 5 days each week. Treatment began between the twenty-fourth and twenty-eighth day of pregnancy (mean duration: 26.3 days), and this dosage schedule was continued without change until delivery. The fetus at delivery was weighed, sacrificed, and a complete autopsy done with appropriate histological sections made. A buccal smear was studied for sex chromatin (Table 1). The second group consists of ten pregnancies in Rhesus monkeys. As soon as the pregnancy was detected, these animals received norethindrone ( 17 a-ethinyl-1 g-nortestosterone) intramuscularly in the form of Norlutin enanthate.” The dose was 25 mg. daily for 5 days each week beginning between the twenty-seventh and thirty-fifth day (mean duration : 31.8 days) of pregnancy. This was continued without change until delivery. The fetus was handled in the identical manner as in Group I and buccal smears for sex chromatin were also studied (Table II). The female monkeys frequently were used
*Parke,
Davis
& Company,
Detroit,
Michigan.
lesions
and
norethindrone
703
for both parts of the study with some animals having several pregnancies. The same male animals were used throughout the study for mating. The same standard diet was used throughout the study, and no other medications of any type were used on any pregnant animal. Results
The results of this study are summarized in Tables I and II. When progesterone was given throughout pregnancy, all animals were delivered at about term (152 to 167 days; mean 160.2 days) living offspring of normal weight (350 to 420 grams; mean 383.4 grams). By chance, in the 6 pregnancies so treated, there were 3 males and 3 females. The male external genitals were entirely normal, and the testes had descended into the scrotum. No abnormalities were noted in any of these male animals at autopsy. The adrenals as well as the other organs were normal. Histological study of the testes showed u ell-developed tubules without spermatogenesis. The prostate, vas, and epididymis were normal and the adrenals likewise showed no microscopic deviation from normal. The 3 female infants of progesteronetreated mothers likewise were entirely normal. The labia, clitoris, vagina, uterus, tubes, and ovaries were of normal size, shape, and position for a newborn. The other organs similarly were normal. Microscopically, the ovary was well developed in each case. There were many primordial ova with some tendency to follicle formation in most ovaries. The cortical stroma showed some fibrous septa with normal-appearing oval or spindle-shaped stromal cells. Around a few follicles there is the suggestion of theta but no evidence of luteinization. A well-formed tunica covered the ovary, and the medulla was quite distinct and, generally, free from ova (Fig. 1) . No hilar cells have been identified. The endometria of these animals vary somewhat in appearance. As in Fig. 2, the endometrial stroma is quite compact and composed of small round cells. The glands
704
Wharton
and
Scott
are simple and tubular. The myometrium is quite thin. Fig. 3 shows considerable progestational response. The endometrium is much thicker. the cells are larger with more abundant cytoplasm approaching a decidual re-
Fig.
1. Progesterone-treated
animal
22B;
ovary.
(x50.)
Fig.
2. Progesterone-treated
animal
22B;
uterus.
(x50.)
action. The endorr retrial glands are tort1 LIOUS, tufted, and lined by fairly low colu~ rmar epithelium similar to that of pregnant ;xdult endometrium. The 3 tubes and vagina were normal. No prosta .tic tissue has been fcjund.
Genital
Fig. 3. Progesterone-treated
lesions
and
norethindrone
705
animal 19B; uterus. (x50.)
and other organs including the adrenal glands were also quite normal. The buccal smear for sex chromatin in each instance corresponded to the proper gonad sex. The 10 pregnancies of the norethindronetreated animals differed markedly from those of Group I. Only 2 progressed to at least 160 days or term, and only 1 additional term-sized fetus (by weight) was delivered at 133 days. The duration of the other 7 pregnancies varied between 96 and 143 days, and the birth weights were compatible with their gestational age, varying between 143 grams and 300 grams. The weight of newborn 23B-2 could not be determined since it was partly eaten, but fortunately the genitourinary tract was spared. The condition of the newborn at delivery was even more unusual. There was only one which was born alive at term (19B), and 1 premature (21B), was also live born. All others were stillborn. Only one (23B-2) was macerated. The 5 newborn males’ external genitals were all normally formed according to their degree of maturity. The phallus was of normal size, and the urethra extended to its end. The scrotal folds had
a normal appearance but were empty. In 3 (16B-1, 18B, and 2OB-1) the testes were in the inguinal canal area. This is a normal finding in many males, but in 2 (16B-2 and 23B-2) the testes were found in the abdominal cavity occupying the positions of the normal ovary. This is not unexpected in such an immature animal as 23B-2 but is rather unusual in the more mature 16B-2. The spermatic cords in the latter 2 animals extended downward toward the prostate which was well defined in all cases. No other gross abnormalities were identified. Histological sections of the testes in each instance showed varying degrees of immaturity commensurate with that of the animal. Tubule formation was generally present. In the more immature animals, the prostate consisted of solid cords of mesenchyma1 condensations about the bladder neck, while in the more mature males, lobulation and tubules were well formed. The adrenals as well as other organs were normal and buccal smears for sex chromatin were negative. The 5 newborn females all were masculinized, and the abnormality in each instance
706
Wharton
Fig.
and
4.
Drawing
Scott
showing
virilized
norethindrone-treated
was the same. From the external appearance of the genitals, they were males (Fig. 4). A phallus of about normal size was present. There were scrotal folds which were empty and bilateral soft inguinal swellings suggesting inguinal testes. The urethral meatus was present at the end of the phallus. On opening the abdomen in each instance, a well-developed female reproductive tract was evident (Fig. 5). The normal-appearing arcuate uterus with delicate tubes and ovaries was present in normal position. The round ligaments were somewhat thickened but were otherwise normal. The uterus then communicated with a rudimentary vagina. This formed a small pocket about the cervix, and its only communication was through a small short midline opening into the posterior urethra immediately below the bladder
female
Macaca
mulatta
fetus.
neck. The urethra then formed a urogenital sinus, and this was patent to the end of the phallus. A condensation of prostatic tissue was present at the bladder neck. All other organs were grossly and histologically normal. There were no seminal vesicles. Buccal smears for sex chromatin were positive (fcmale) in each instance. The histology of the ovaries in these animals is most interesting and varied. Figs. 6> 7, and 8 represent the findings in the more mature animals. The ovaries have a well defined tunica, cortex, and medulla. The primordial ova lie in the cortex surrounded by a dense compact stroma. Some fibrous septa are present. The hilar area is quite vascular and generally the medullary portion is composed of a fairly loose fibrous stroma. Occasionally, as seen in Fig. 7, there
Genital
Fig. 5. Norethindrone-treated communication with urethra
Figs. 6 through Fig. 6. Animal
animal 19B, is demonstrated.
17. Norethindrone-treated 19B;
ovary.
(x50.)
bladder
animals.
and
urethra
opened.
lesions
Vaginal
and
norethindrone
707
708
Wharton
and
Scott
are cordlike cellular condensations in the medulla. These are cortical stromal cells which have not as yet reached the cortex. No follicles have formed and no granulosa can be identified. With these exceptions
Fig. 7. Animal
Fig. 8. Animal
31B;
23B-1;
ov:~ry.
ovary.
( 50.)
(. 50.)
to those ! 01 these ovaries are quite similar animals or any the progesterone :-treated other normal ne\ Yborn female Rhesus. On the other extreme, however, arc thr: WXi ovaries of anima ,l 23B-1. This animal
Genital
rathe :r premature, being delivered at 128 clays and weighing 280 g;rams. The ovaries are E:ven more immature than this, and the right differs greatly fron 1 the left. Figs. 8 an cl 9 show one ox-ary. The cortical con-
Fig.
9. Animal
Fig.
10. Animal
23B-1;
lesions
norethindrone
709
densation is evident and distinct from the medulla but contains very few primordial ova. There is a suggestion that the tunica is forming, but generally the cortex is composed of cords of fairly small dark cells with
ovary.
(~200.)
^S
am.dw.
23B-1;
and
opposite
ovary
to Fig.
9. (x50.)
710
Wharton
and Scott
fairly broad fibrous septa. These cords resemble in some degree the undifferentiated epithelial cords of the primitive testis OI even more the cordlike arrangement in some arrhenoblastomas. The rete is quite vascu-
Fig.
11. Animal
Fig. 12. Control
23B-1;
ovary
ovary
in Fig.
at 118 days.
(x50.)
10.
(x200.)
lar, and there are a the loose fibrous ma The opposite ovar even more immatur fact it appears to
few cords but no ova in tris of the medulla. ‘y in the same animal is e (Figs. 10 and 11 I. in bc cornpletelv discqa-
Genital
nixed. There is a sugge tion 01rerlying a poorly cortex. In this cortex entiate d epithelial cell fibrous septa. In man
stion of tunica formaformed, thin primary are cords of undiffers, few ova, and thin y instances the cords
Fig. 13. Control
Fig. 14. Animal
ovary
23B-1;
lesions
and
norethindrone
711
are poorly formed and have a rather jumbled appearance. There are hilar vessels, and the medulla is composed of a disorganized conglomeration of undifferentiated sex cords, a few ova, and a loose fibrous
at 109 days.
prostate.
(x50.)
(x50.)
712
Wharton
and
Scott
matrix. This pattern is similar to that seen in the more anaplastic forms of arrhenohlastoma, and there is very little that would resemble normal ovarian tissue. It is, in fact, an undifferentiated gonad.
Fig.
15. Animal
19B;
endometrium.
Fig.
16. Animal
19R; endometrirun.
(x50.)
(~200.)
For comparison the two ovaries 01 animal may be COInpared to those of era1 untreated and. , hence, control an which we were fc lrtunate to obtain the Carnegie Instit ute of EmbryoloLgy.
Volun1c Number
89 6
Genital
Fig.
17. Animal
23B; endometrium.
ovary (Fig. 12) was from a premature Rhesus delivered at 118 days’ gestation. The other (Fig. 13) is from a 109 day pregnancy. Both of these ovaries show more maturation and better development than those of animal 22B-1, although one is 10 days younger and the other 19 days more premature. In addition to the histologic changes in the ovaries, each of the 5 masculinized females had well-defined prostatic tissue at the bladder neck in the region where it normally would be found in the males. These prostates tended to have a lobular pattern surrounded by a capsule. There are cords of epithelial cells which for the most part are solid. In some areas tubules are forming, making a rudimentary glandular pattern (Fig. 14) which is supported by a developing connective tissue matrix. The uterus was normally formed in each instance with a normal cervix, myometrium, and endometrial cavity. The endometria of these animals showed evidence generally of the marked hormonal stimulation. No decidua was encountered; however, the endometrirlm frequently was quite thick with
lesions
and
norethindrone
713
(x50.)
rather large tortuous glands (Fig. 15) lined by tall tufted secretory columnar epithelium. The stroma generally was fairly compact, but the cells were of moderate size (Fig. 16) and contained a moderate amount of cytoplasm. In other animals the endometrial response is Iess marked. It is thinner and composed of simple tubular glands lined by low columnar to almost cuboidal epithelium (Fig. 17). The stroma is composed of relatively small closely packed cells. The Fallopian tubes are quite normal histologically, and the vaginal mucosa is relatively thick showing the usual normal degree of keratinization found at birth. The urethra is quite normal. The inguinal areas show only loose areolar connective tissue. All other organs are normal histologicall) and compatible with any other Rhesus monkey of comparable development. Comment
The interpretation of the above observations is another matter. Progesterone in comparatively large doses requires no further discussion. It produces no anomalies and does not interfere with the normal con-
714
Wharton
and
Scott
elusion of a pregnancy in the Macaca muZatta. In short these animals are the same as any untreated animal in the colony and this serves as a double control. In regard to norethindrone, it appears to have a marked androgenic effect on the fetus comparable, indeed, to testosterone yet no demonstrable androgenic effect has been noted in the mother who has actually received the intramuscular injections. From this one may conclude that the developing genitals of the fetus are much more sensitive to androgenic stimulation than those in the mature animal. Testosterone and norethindrone produce similar effects. There seems to be no need of invoking any, maternal metabolic abnormality nor a difference in placental transmission. There is nothing to suggest further that the fetus metabolizes these substances differently. The fetal adrenal glands are proportionately immature in the immature newborn and appear to mature normally as other animals arc studied. The ovary on the other hand at times appears to be disproportionately retarded in regard to its maturation process. This is most notable in animal 23B-1. Norethindrone was started on day 32 of pregnancy, and the animal was delivered of a stillborn masculinized fetus weighing 280 grams at 128 days (Figs. 8 through 11). This change is not predictable since animal 21B was started on norethindrone on day 35 of pregnancy, or 3 days later, and was delivered of a living masculinized 300 gram fetus at 143 days. There seems to be a much greater than normal degree of retardation in animal 23B-1 than in 21R (Fig. 7) despite the 20 gram weight differential and the 15 additional days of pregnancy in the latter. We were not too surprised that all 5 female fetuses were masculinized. The extent of the than those changes, however, are greater encountered clinically by Wilkins” and Jacobson.:’ Their findings generally were only clitoral enlargement or enlargement of the clitoris with labioscrotal fusion. The rudimentary vagina ending blindly with only a small opening in the posterior urethra was
July 15. 1964 Am. J. Obst. & Gyner.
not reported in these cases, and the presence or absence of prostatic tissue is not known since, presumably, few if any of these children had laparotomies. These animals showed no evidence of adrenal hyperplasia or other histological abnormality. This does not preclude any aberration of function, yet there is nothing to support this possibility. The fact that only 2 of 10 fetuses of the norethindrone-treated adults were born alive and that only 3 were of term weight is quite unexpected. Usually progestins might be expected to behave, at least in some respects. as progesterone, and one would normally predict that they might prevent abortion or premature delivery. The fact is that norrthindrone actually appears lo induce premature labor and probably fetal death in utero since 8 of 10 were stillborn and one of these was moderately macerated. The mechanism of this is not certain. It is possible that the relatively large amount of the administered drug (25 mg. a day for 5 days each week) is the responsible factor. These monkeys have the unfortunate habit of eating the placenta immediately after delivery an d in no instance did we have the good fortune to obtain a placenta for study. No endocrine studies have been done on these pregnant animals thus far. There are not to our knowledge even sufficient normal baseline studies for comparison. Basic information such as this is needed before the mechanism of premature delivery in these animals can be understood. The most likely possibility would seem to be suppression of placental function in some way, but further speculation would be pure conjecture. It is interesting that norethindrone at a comparably much lower dose level and by mouth does not appear to have an adverse effect on the duration of pregnancy in the human. The reason for the variance between Homo sa$?ns and Macaca mulatta is not clear, but in both species norethindrone ohviously has some androgenic potential. The only clinical observation that can be made from this study thus far is that norethindrone may have dangerous potentials when given to pregnant women.
Volume Number
a9 6
Genital
Summary
Macaca mulatta were
given
either progesterone or norethindrone beginning in early pregnancy and continuing until delivery. The findings in this study to date are unequivocal and clear. These are: 1. Progesterone in the dosage used produced no change in the duration of the pregnancy nor did it adversely affect the survival of the fetus. 2. Progesterone did not produce any anomalies of either the reproductive tract or any other organ in the newborn Rhesus. 3. Norethindrone exerts a deleterious effect on pregnancy in the Rhesus monkey with only 2 pregnancies progressing to term
lesions
and
norethindrone
715
by dates and only one additional animal being of term size at delivery. 4. The effects of norethindrone on the fetus are catastrophic. Eight of 10 were stillborn, and all 5 female infants showed uniform and marked virilization affecting primarily the external genitals. The clitoris was enlarged with the urethra extending to its end while the vagina was rudimentary entering the posterior urethra near the vesical sphincter. Prostatic tissue was formed and the development of the ovary appeared to be retarded. 5. In the norethindrone treated male newborn, cryptorchidism was uniform and in 2 animals the testes lay within the abdominal cavity.
REFERENCES
1. Wilkins, L., Jones, H. W., Jr., Holman, G. H., and Stempfel, R. S., Jr.: J. Clin. Endocrinol. 18: 559, 1958. 2. Wilkins, L.: J. A. M. A. 172: 1028, 1960. 3. Jacobson, B. J.: AM. J. OBST. & GYNEC. 84: 962, 1962.
4.
Wells, L. J., and VanWagenen, G.: Contrib. Embryol. 35: 93, 1954. 5. Corner, G. W.: Contrib. Embryo]. 15: 73, 1923. 6. Schultz, A. H.: Carnegie Inst. of Washington Pub. 538, 1941.
89
number
6
July 15, 1964
American
Journal
of Obstetrics and Gynecology
GYNECOLOGY Experimental production of genital lesions with norethindrone LAWRENCE ROGER Baltimore,
R. B.
SCOTT,
WHARTON,
JR.,
M.D.
M.D.
Maryland
T H E L A T E Lawson Wilkins and his associates’! 2 in 1958 and again in 1960 reported a series of masculinized infants with apparently normally functioning adrenal glands. The important factor in the production of these anomalies appeared to be the treatment of the pregnant mother with an assortment of hormones. Among 101 cases collected by Wilkins, 34 had received ethister-
one; 35, norethindrone; I, norethynodrel ; 15, testosterone; 2, progesterone; 4, stilbestrol; and 10, no endocrine therapy. In each female infant, the clitoris was enlarged and there were varying degrees of labioscrotal fusion. The infants were normal in all other respects and became normally feminized later with normal menarche, menstruation, and ovulation. All thlls far investigated ha1.e had chromatin-positive buccal smears. In 1962 Jacobson:, treated 385 consecutive pregnant private patients with norethindrone noting a 5.5 per cent incidence of maternal virilization and an 18.3 per cent over-all incidence of fetal virilization. The frequency of these changes rose to 23.7 per cent when norethindrone was started by the twelfth week of pregnancy. Because of the findings of Wilkins and his group our interest was aroused in this subject. The fact that androgens given to pregnant animals can virilize their offspring had been well established in a variety of ani-
From the Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, and the Baltimore, Maryland, and the Department of Obstetrics and Gynecology, Western Reserve Unirlersity School of Medicine, Cleveland, Ohio. This work was supported in part by Research Grant No. AM 0592% of the National Institutes of Health, Nation Institute of Arthritis and Metabolic Diseases. Progesterone in oil was generousl) supplied by Ayerst Laboratories. Norethindrone was generously supplied as No&tin by Parke, Davis B Company.
701
702
Wharton
Table
and
I. Progesterone
Animal 18B 19B 2OB
study Duration pregnancy 167 158 158
Progesterone started (day) 26 24 28 28 ‘8 2-P .__
21B 22B 23B
Table
July 1.5, 1')h.l Am. J. Ohst. B (;ynw.
Scott
II. Norethindrone
Condition at birth
(grams)
152 161 165
Sex
1 Buccal smear chromatin
350 350 400
Living Living Living
0 i
i-+ .
420 400 400
Living Living Living
8 P 8
t -
study i :[I;:
i dr%+;;;;:;ed
___-
--___Birth weight
of
( ii;;)
1 C$X;~--~
1
1 ciE;in
Remarks
--
Animal 32 33 35 31 32 27 35 27 79 2
16B-1 16B-2 18B 19B ?OB-1 “OB-2 21B 22B 23B-1 23B-2
128 131 162 167 133 111 143 96
260 300 550 450 400 300 300 143
128
280
104
mals. Most pertinent to this study was the work of Wells and VanWagenen.4 They treated pregnant Rhesus monkeys with testosterone proprionate in oil beginning on the forty-first to sixty-ninth day of pregnancy and continuing through the ninetyninth day. Two untreated controls were normal, but 9 of 10 treated animals were virilized. The findings consisted of an elongated urogenital sinus and absence of the distal vagina but prostatic tissue and seminal vesicles were found. These changes persisted into adult life, but the animals had a normal menarche and menses. Material
and
methods
With this background information the study was started using the Macaca mulatta initially in the animal colony of the Carnegie Institute of Embryology and later at the Primate Center of the Johns Hopkins University. Personnel5 working with the Carnegie Institute of Embryology animal colony have long been interested in breeding Rhesus monkeys and records are available of many
Stillborn Stillborn Stillborn Living Stillborn Stillborn Living Stillborn Stillborn Macerated
i 8 ; 9 i 0 0
: + + + +
Inguinal testes Intra-abdominal testes Inguinal testes Masculinized Jnguinal testes Masculinized Masculinized Intra-abdominal testes Masculinized Masculinized -.---.
pregnancies which may in a way serve as controls. These animals usually have approximately a 28 day menstrual cycle with ovulation usually occurring about 15 days prior to menstruation. The average duration of a normal gestation is considered to be 160 to 165 days as estimated from ovulation rather than from the preceding menstrual period. The pregnancies usually proceed uneventfully to term, and abortion or stillbirth is unusual. The parturient monkey rapidly eats the placenta making this or,qan usually unavailable for study. The newborn term Rhesus monkey weighs in the nei,ghborhood of 100 grams’ and has well-developed genitals. Usually it is quite easy to distinguish between male and female newborn. Occasionally at birth the testes are undescended, lying in the inguinal area rather than in the scrotum. There are no other genital variants and spontaneous malformations of the reproductive tract have not been observed in this colony. The uterus is normal arcuate in type. Mature female Macaca mulattas, after
Volume Number
89 6
Genital
being received in the colony, were held for a period of observation during which time an attempt was made to correct nutritional deficiencies, improve their general health, and record the character of their menstrual cycles. After this had been accomplished, the animals were mated for usually a period of 4 to 5 days beginning on day 9 or 10 of the menstrual cycle. These animals have a cycle of about 28 days’ duration and frequently do not develop amenorrhea until the second menstrual period following fertilization. For this reason internal genital organs of the animals were palpated following each menstrual period or whenever the cycle was missed. As soon as the pregnancy could be diagnosed by palpation, hormone injections were commenced. In the first part of the study 6 pregnant Rhesus monkeys were treated with progesterone in oil intramuscularly, receiving 50 mg. daily for 5 days each week. Treatment began between the twenty-fourth and twenty-eighth day of pregnancy (mean duration: 26.3 days), and this dosage schedule was continued without change until delivery. The fetus at delivery was weighed, sacrificed, and a complete autopsy done with appropriate histological sections made. A buccal smear was studied for sex chromatin (Table 1). The second group consists of ten pregnancies in Rhesus monkeys. As soon as the pregnancy was detected, these animals received norethindrone ( 17 a-ethinyl-1 g-nortestosterone) intramuscularly in the form of Norlutin enanthate.” The dose was 25 mg. daily for 5 days each week beginning between the twenty-seventh and thirty-fifth day (mean duration : 31.8 days) of pregnancy. This was continued without change until delivery. The fetus was handled in the identical manner as in Group I and buccal smears for sex chromatin were also studied (Table II). The female monkeys frequently were used
*Parke,
Davis
& Company,
Detroit,
Michigan.
lesions
and
norethindrone
703
for both parts of the study with some animals having several pregnancies. The same male animals were used throughout the study for mating. The same standard diet was used throughout the study, and no other medications of any type were used on any pregnant animal. Results
The results of this study are summarized in Tables I and II. When progesterone was given throughout pregnancy, all animals were delivered at about term (152 to 167 days; mean 160.2 days) living offspring of normal weight (350 to 420 grams; mean 383.4 grams). By chance, in the 6 pregnancies so treated, there were 3 males and 3 females. The male external genitals were entirely normal, and the testes had descended into the scrotum. No abnormalities were noted in any of these male animals at autopsy. The adrenals as well as the other organs were normal. Histological study of the testes showed u ell-developed tubules without spermatogenesis. The prostate, vas, and epididymis were normal and the adrenals likewise showed no microscopic deviation from normal. The 3 female infants of progesteronetreated mothers likewise were entirely normal. The labia, clitoris, vagina, uterus, tubes, and ovaries were of normal size, shape, and position for a newborn. The other organs similarly were normal. Microscopically, the ovary was well developed in each case. There were many primordial ova with some tendency to follicle formation in most ovaries. The cortical stroma showed some fibrous septa with normal-appearing oval or spindle-shaped stromal cells. Around a few follicles there is the suggestion of theta but no evidence of luteinization. A well-formed tunica covered the ovary, and the medulla was quite distinct and, generally, free from ova (Fig. 1) . No hilar cells have been identified. The endometria of these animals vary somewhat in appearance. As in Fig. 2, the endometrial stroma is quite compact and composed of small round cells. The glands
704
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are simple and tubular. The myometrium is quite thin. Fig. 3 shows considerable progestational response. The endometrium is much thicker. the cells are larger with more abundant cytoplasm approaching a decidual re-
Fig.
1. Progesterone-treated
animal
22B;
ovary.
(x50.)
Fig.
2. Progesterone-treated
animal
22B;
uterus.
(x50.)
action. The endorr retrial glands are tort1 LIOUS, tufted, and lined by fairly low colu~ rmar epithelium similar to that of pregnant ;xdult endometrium. The 3 tubes and vagina were normal. No prosta .tic tissue has been fcjund.
Genital
Fig. 3. Progesterone-treated
lesions
and
norethindrone
705
animal 19B; uterus. (x50.)
and other organs including the adrenal glands were also quite normal. The buccal smear for sex chromatin in each instance corresponded to the proper gonad sex. The 10 pregnancies of the norethindronetreated animals differed markedly from those of Group I. Only 2 progressed to at least 160 days or term, and only 1 additional term-sized fetus (by weight) was delivered at 133 days. The duration of the other 7 pregnancies varied between 96 and 143 days, and the birth weights were compatible with their gestational age, varying between 143 grams and 300 grams. The weight of newborn 23B-2 could not be determined since it was partly eaten, but fortunately the genitourinary tract was spared. The condition of the newborn at delivery was even more unusual. There was only one which was born alive at term (19B), and 1 premature (21B), was also live born. All others were stillborn. Only one (23B-2) was macerated. The 5 newborn males’ external genitals were all normally formed according to their degree of maturity. The phallus was of normal size, and the urethra extended to its end. The scrotal folds had
a normal appearance but were empty. In 3 (16B-1, 18B, and 2OB-1) the testes were in the inguinal canal area. This is a normal finding in many males, but in 2 (16B-2 and 23B-2) the testes were found in the abdominal cavity occupying the positions of the normal ovary. This is not unexpected in such an immature animal as 23B-2 but is rather unusual in the more mature 16B-2. The spermatic cords in the latter 2 animals extended downward toward the prostate which was well defined in all cases. No other gross abnormalities were identified. Histological sections of the testes in each instance showed varying degrees of immaturity commensurate with that of the animal. Tubule formation was generally present. In the more immature animals, the prostate consisted of solid cords of mesenchyma1 condensations about the bladder neck, while in the more mature males, lobulation and tubules were well formed. The adrenals as well as other organs were normal and buccal smears for sex chromatin were negative. The 5 newborn females all were masculinized, and the abnormality in each instance
706
Wharton
Fig.
and
4.
Drawing
Scott
showing
virilized
norethindrone-treated
was the same. From the external appearance of the genitals, they were males (Fig. 4). A phallus of about normal size was present. There were scrotal folds which were empty and bilateral soft inguinal swellings suggesting inguinal testes. The urethral meatus was present at the end of the phallus. On opening the abdomen in each instance, a well-developed female reproductive tract was evident (Fig. 5). The normal-appearing arcuate uterus with delicate tubes and ovaries was present in normal position. The round ligaments were somewhat thickened but were otherwise normal. The uterus then communicated with a rudimentary vagina. This formed a small pocket about the cervix, and its only communication was through a small short midline opening into the posterior urethra immediately below the bladder
female
Macaca
mulatta
fetus.
neck. The urethra then formed a urogenital sinus, and this was patent to the end of the phallus. A condensation of prostatic tissue was present at the bladder neck. All other organs were grossly and histologically normal. There were no seminal vesicles. Buccal smears for sex chromatin were positive (fcmale) in each instance. The histology of the ovaries in these animals is most interesting and varied. Figs. 6> 7, and 8 represent the findings in the more mature animals. The ovaries have a well defined tunica, cortex, and medulla. The primordial ova lie in the cortex surrounded by a dense compact stroma. Some fibrous septa are present. The hilar area is quite vascular and generally the medullary portion is composed of a fairly loose fibrous stroma. Occasionally, as seen in Fig. 7, there
Genital
Fig. 5. Norethindrone-treated communication with urethra
Figs. 6 through Fig. 6. Animal
animal 19B, is demonstrated.
17. Norethindrone-treated 19B;
ovary.
(x50.)
bladder
animals.
and
urethra
opened.
lesions
Vaginal
and
norethindrone
707
708
Wharton
and
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are cordlike cellular condensations in the medulla. These are cortical stromal cells which have not as yet reached the cortex. No follicles have formed and no granulosa can be identified. With these exceptions
Fig. 7. Animal
Fig. 8. Animal
31B;
23B-1;
ov:~ry.
ovary.
( 50.)
(. 50.)
to those ! 01 these ovaries are quite similar animals or any the progesterone :-treated other normal ne\ Yborn female Rhesus. On the other extreme, however, arc thr: WXi ovaries of anima ,l 23B-1. This animal
Genital
rathe :r premature, being delivered at 128 clays and weighing 280 g;rams. The ovaries are E:ven more immature than this, and the right differs greatly fron 1 the left. Figs. 8 an cl 9 show one ox-ary. The cortical con-
Fig.
9. Animal
Fig.
10. Animal
23B-1;
lesions
norethindrone
709
densation is evident and distinct from the medulla but contains very few primordial ova. There is a suggestion that the tunica is forming, but generally the cortex is composed of cords of fairly small dark cells with
ovary.
(~200.)
^S
am.dw.
23B-1;
and
opposite
ovary
to Fig.
9. (x50.)
710
Wharton
and Scott
fairly broad fibrous septa. These cords resemble in some degree the undifferentiated epithelial cords of the primitive testis OI even more the cordlike arrangement in some arrhenoblastomas. The rete is quite vascu-
Fig.
11. Animal
Fig. 12. Control
23B-1;
ovary
ovary
in Fig.
at 118 days.
(x50.)
10.
(x200.)
lar, and there are a the loose fibrous ma The opposite ovar even more immatur fact it appears to
few cords but no ova in tris of the medulla. ‘y in the same animal is e (Figs. 10 and 11 I. in bc cornpletelv discqa-
Genital
nixed. There is a sugge tion 01rerlying a poorly cortex. In this cortex entiate d epithelial cell fibrous septa. In man
stion of tunica formaformed, thin primary are cords of undiffers, few ova, and thin y instances the cords
Fig. 13. Control
Fig. 14. Animal
ovary
23B-1;
lesions
and
norethindrone
711
are poorly formed and have a rather jumbled appearance. There are hilar vessels, and the medulla is composed of a disorganized conglomeration of undifferentiated sex cords, a few ova, and a loose fibrous
at 109 days.
prostate.
(x50.)
(x50.)
712
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matrix. This pattern is similar to that seen in the more anaplastic forms of arrhenohlastoma, and there is very little that would resemble normal ovarian tissue. It is, in fact, an undifferentiated gonad.
Fig.
15. Animal
19B;
endometrium.
Fig.
16. Animal
19R; endometrirun.
(x50.)
(~200.)
For comparison the two ovaries 01 animal may be COInpared to those of era1 untreated and. , hence, control an which we were fc lrtunate to obtain the Carnegie Instit ute of EmbryoloLgy.
Volun1c Number
89 6
Genital
Fig.
17. Animal
23B; endometrium.
ovary (Fig. 12) was from a premature Rhesus delivered at 118 days’ gestation. The other (Fig. 13) is from a 109 day pregnancy. Both of these ovaries show more maturation and better development than those of animal 22B-1, although one is 10 days younger and the other 19 days more premature. In addition to the histologic changes in the ovaries, each of the 5 masculinized females had well-defined prostatic tissue at the bladder neck in the region where it normally would be found in the males. These prostates tended to have a lobular pattern surrounded by a capsule. There are cords of epithelial cells which for the most part are solid. In some areas tubules are forming, making a rudimentary glandular pattern (Fig. 14) which is supported by a developing connective tissue matrix. The uterus was normally formed in each instance with a normal cervix, myometrium, and endometrial cavity. The endometria of these animals showed evidence generally of the marked hormonal stimulation. No decidua was encountered; however, the endometrirlm frequently was quite thick with
lesions
and
norethindrone
713
(x50.)
rather large tortuous glands (Fig. 15) lined by tall tufted secretory columnar epithelium. The stroma generally was fairly compact, but the cells were of moderate size (Fig. 16) and contained a moderate amount of cytoplasm. In other animals the endometrial response is Iess marked. It is thinner and composed of simple tubular glands lined by low columnar to almost cuboidal epithelium (Fig. 17). The stroma is composed of relatively small closely packed cells. The Fallopian tubes are quite normal histologically, and the vaginal mucosa is relatively thick showing the usual normal degree of keratinization found at birth. The urethra is quite normal. The inguinal areas show only loose areolar connective tissue. All other organs are normal histologicall) and compatible with any other Rhesus monkey of comparable development. Comment
The interpretation of the above observations is another matter. Progesterone in comparatively large doses requires no further discussion. It produces no anomalies and does not interfere with the normal con-
714
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and
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elusion of a pregnancy in the Macaca muZatta. In short these animals are the same as any untreated animal in the colony and this serves as a double control. In regard to norethindrone, it appears to have a marked androgenic effect on the fetus comparable, indeed, to testosterone yet no demonstrable androgenic effect has been noted in the mother who has actually received the intramuscular injections. From this one may conclude that the developing genitals of the fetus are much more sensitive to androgenic stimulation than those in the mature animal. Testosterone and norethindrone produce similar effects. There seems to be no need of invoking any, maternal metabolic abnormality nor a difference in placental transmission. There is nothing to suggest further that the fetus metabolizes these substances differently. The fetal adrenal glands are proportionately immature in the immature newborn and appear to mature normally as other animals arc studied. The ovary on the other hand at times appears to be disproportionately retarded in regard to its maturation process. This is most notable in animal 23B-1. Norethindrone was started on day 32 of pregnancy, and the animal was delivered of a stillborn masculinized fetus weighing 280 grams at 128 days (Figs. 8 through 11). This change is not predictable since animal 21B was started on norethindrone on day 35 of pregnancy, or 3 days later, and was delivered of a living masculinized 300 gram fetus at 143 days. There seems to be a much greater than normal degree of retardation in animal 23B-1 than in 21R (Fig. 7) despite the 20 gram weight differential and the 15 additional days of pregnancy in the latter. We were not too surprised that all 5 female fetuses were masculinized. The extent of the than those changes, however, are greater encountered clinically by Wilkins” and Jacobson.:’ Their findings generally were only clitoral enlargement or enlargement of the clitoris with labioscrotal fusion. The rudimentary vagina ending blindly with only a small opening in the posterior urethra was
July 15. 1964 Am. J. Obst. & Gyner.
not reported in these cases, and the presence or absence of prostatic tissue is not known since, presumably, few if any of these children had laparotomies. These animals showed no evidence of adrenal hyperplasia or other histological abnormality. This does not preclude any aberration of function, yet there is nothing to support this possibility. The fact that only 2 of 10 fetuses of the norethindrone-treated adults were born alive and that only 3 were of term weight is quite unexpected. Usually progestins might be expected to behave, at least in some respects. as progesterone, and one would normally predict that they might prevent abortion or premature delivery. The fact is that norrthindrone actually appears lo induce premature labor and probably fetal death in utero since 8 of 10 were stillborn and one of these was moderately macerated. The mechanism of this is not certain. It is possible that the relatively large amount of the administered drug (25 mg. a day for 5 days each week) is the responsible factor. These monkeys have the unfortunate habit of eating the placenta immediately after delivery an d in no instance did we have the good fortune to obtain a placenta for study. No endocrine studies have been done on these pregnant animals thus far. There are not to our knowledge even sufficient normal baseline studies for comparison. Basic information such as this is needed before the mechanism of premature delivery in these animals can be understood. The most likely possibility would seem to be suppression of placental function in some way, but further speculation would be pure conjecture. It is interesting that norethindrone at a comparably much lower dose level and by mouth does not appear to have an adverse effect on the duration of pregnancy in the human. The reason for the variance between Homo sa$?ns and Macaca mulatta is not clear, but in both species norethindrone ohviously has some androgenic potential. The only clinical observation that can be made from this study thus far is that norethindrone may have dangerous potentials when given to pregnant women.
Volume Number
a9 6
Genital
Summary
Macaca mulatta were
given
either progesterone or norethindrone beginning in early pregnancy and continuing until delivery. The findings in this study to date are unequivocal and clear. These are: 1. Progesterone in the dosage used produced no change in the duration of the pregnancy nor did it adversely affect the survival of the fetus. 2. Progesterone did not produce any anomalies of either the reproductive tract or any other organ in the newborn Rhesus. 3. Norethindrone exerts a deleterious effect on pregnancy in the Rhesus monkey with only 2 pregnancies progressing to term
lesions
and
norethindrone
715
by dates and only one additional animal being of term size at delivery. 4. The effects of norethindrone on the fetus are catastrophic. Eight of 10 were stillborn, and all 5 female infants showed uniform and marked virilization affecting primarily the external genitals. The clitoris was enlarged with the urethra extending to its end while the vagina was rudimentary entering the posterior urethra near the vesical sphincter. Prostatic tissue was formed and the development of the ovary appeared to be retarded. 5. In the norethindrone treated male newborn, cryptorchidism was uniform and in 2 animals the testes lay within the abdominal cavity.
REFERENCES
1. Wilkins, L., Jones, H. W., Jr., Holman, G. H., and Stempfel, R. S., Jr.: J. Clin. Endocrinol. 18: 559, 1958. 2. Wilkins, L.: J. A. M. A. 172: 1028, 1960. 3. Jacobson, B. J.: AM. J. OBST. & GYNEC. 84: 962, 1962.
4.
Wells, L. J., and VanWagenen, G.: Contrib. Embryol. 35: 93, 1954. 5. Corner, G. W.: Contrib. Embryo]. 15: 73, 1923. 6. Schultz, A. H.: Carnegie Inst. of Washington Pub. 538, 1941.