The Effects of Androgens on Fetal Sexual Development. Androgen-induced Female Pseudohermaphrodism

Transcription

1 The Effects of Androgens on Fetal Sexual Development Androgen-induced Female Pseudohermaphrodism Melvin M. Grumbach, M.D., and Jacques R. Ducharme, M.D. IN ANY CONSIDERATION of the effects of androgens on the fetal organism, it is important that the term "androgen" be defined. An androgen is usually regarded as a steroid which, in the mature male animal, is capable of compensating for the effects of castration and which, in the immature animal, stimulates the development of male accessory sex organs and secondary sex characteristics; it may also be defined as a substance which, in the fetus, promotes masculinization of the primordial genital tract. Any assessment of the androgenic properties of a substance must take into account the maturity of the organism. It is well known that the potency of an androgen may vary with the route of administration, the responsiveness of the end-organ, and the species (and even the strain) of assay animal. 1 Not as well appreciated is the fact that androgenic activity may vary with the stage of development of the organism. For example, in the adult rat, androstenediol is several times as androgenic as methylandrostenediop 3 In contrast, methylandrostenediol is appreciably more potent than andro- ~ From the Department of Pediatrics, College of Physicians & Surgeons, Columbia University, and Babies Hospital, New York, N.Y. Presented at a symposium on "The Effect on Fetal Developmmt of Steroids Given During Pregnancy" at the fifteenth annual meeting of The American Society for the Study of Sterility, Atlantic City, N.J., April4, The work reported in this paper was supported by a research grant from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service, Bethesda, Md. We are grateful to Dr. Joseph W. Jailer for permission to study and report the case described in the appendix to this paper. 157

2 158 GRUMBACH & DUCHARME Fertility & Sterility stenediol in its action on the female fetus of the pregnant raty An androgen may exhibit biological activity unrelated to its primary androgenic action. Methyltestosterone, and indeed testosterone itself have a progestational effect on the estrogen-primed enclometrium G The effect on the fetus, as well as the progestational effect, must be taken into account in assessing the net effects of the administration during pregnancy of certain semisynthetic testosterone analogues with potent oral progestational properies. 7 8 Androgenic substances have not been shown to have a deleterious effect on the development of the genital tract of the male human fetus. The masculinizing morphogenetic secretion of the fetal testis plays a primary role in differentiation of the accessory sex structures Although the effects of synthetic androgens on these structures are similar in many ways to that of the "androgenic" fetal testicular secretion, there is an important difference: The ability of the fetal testis to inhibit the development of the MUllerian ducts is not duplicated in experimental placental mammals by the known steroid androgens EXPERIMENTAL FEMALE PSEUDOHERMAPHRODISM Following the synthesis of testosterone in 1935, investigations were initiated in a number of laboratories to determine its effect on embryonic sexual development, and to test the dihormonal theory of sex differentiation proposed by Lillie in These experiments have been reviewed in detail by Burns/ 2 Gallien/ 3 Jost, 9 14 Moore/" and Wells. 1 G Only experimental female pseudohermaphrodism in placental mammals need concern us here. Table 1 lists some placental mammals in which abnormalities in the female fetus, following antenatal administration of various androgens, have been studied. In most experiments the androgen was administered to the mother; in a few instances, the androgen was injected into the fetus or the amniotic sac. Briefly, effects on the female fetus can be summarized as follows: 1. The urogenital sinus and its derivatives (such as the prostate) and the external genitalia were masculinized. 2. Except in the hamster, 17 1 R the Wolffian ducts did not regress and formed seminal vesicles, epididymides, and to a variable degree, vasa deferentia. 3. In general, development of the Miillerian ducts was not appreciably

3 TABLE 1. Some Comparative Studies of the Masculinizing Effects of Antenatal Administration of Androgens on the Female Fetus of Placental Mammals Order Species Androgen Author Date Rodentia Guinea pig Testosterone propionate Dantchakoff Rat Testosterone propionate Greene, Burrill, Ivy Hamilton and Gardner 1937 Testosterone Greene, Burrill, Ivy Androstenedione Androsterone Dehydroepiandrosterone Androstenediol dipropionate Jost 1953 Methylandrostenediol dipropionate 9a-fluoro-11,80H -17-methyltestosterone Jost 1958 Mouse Testosterone propionate Raynaud Turner Dehydroepiandrosterone Raynaud 1938 Hamster Testosterone propionate Bruner and Witschi 1946 White 1949 lnsectivora Hedgehog Testosterone propionate Mombaerts 1944 Godet 1951 Mole Testosterone propionate Godet 1946,1949 Lagomorpha Rabbit Methyltestosterone Testosterone Methyltestosterone Jost 1945, ethynyltestosterone Adrenosterone Jost a-fluoro-11,80H -17-methyltestosterone Jost 1958 Artiodactyla Cattle Testosterone Mason etal Goat Testosterone Barone Primate Monkey Testosterone propionate Van Wagenen and Hamilton 1943 Dantchakoff 1950 Wells and Van Wagenen 1954

4 160 GRUMBACH & DUCHARME Fertility & Sterility affected, although in some species the development of the caudal part was inhibited.* 4. Inversion of ovarian development was not observed. These experiments also provide a substantial body of indirect evidence for the placental transfer of a number of androgenic steroids in a variety of species. There are species differences in the anatomy of the genital tract as well as in the sensitivity of embryonic end-organs to androgens. Nonetheless, experimental female pseudohermaphrodism has provided useful information about factors which influence the response of the primitive genital tract to androgen. Dosage and Fetal Age The degree of masculinization of the female fetus can be correlated with the dose and type of androgen administered to the pregnant female and with the stage of pregnancy when treatment was begun Burrill and Greene 20 showed that a maximal degree of masculinization could be produced in the female rat fetus with a total dose of 5 mg. of testosterone propionate if injections were begun before the sixteenth day of pregnancy. Ten times this dose did not produce a greater degree of virilization. It was also demonstrated that the potency of testosterone propionate by injection was approximately 8 times that of testosterone, 17 times that of androstenedione, 20 times that of androsterone, and 35 times that of dehydroepiandrosterone. The parts of the genital tract vary in their sensitivity to androgen. The lower genital tract (the external genitalia and urogenital sinus) has a lower threshold of response than the Wolffian ducts; Bnmer and WitschP 7 have considered in detail this caudad-cephalad gradient of sensitivity to androgen. In the rat a difference also has been observed in the responsiveness of the right and left Wolffian ducts. 23 The stage in pregnancy when treatment with androgens is initiated is an essential factor in determining the response of the fetal genital tract. The important principle of the limited period of sensivity of the genital ducts and urogenital sinus to androgens has been emphasized by J ost and convincingly demonstrated in fetal castration experiments. 9 The capacity to stimulate development of the Wolffian duct, masculine derivatives of the * However, Burns,12 and Moore15 observed a remarkable stimulation in the opossum, a nonplacental mammal, of the derivatives of the Mullerian ducts by large doses of testosterone propionate.

5 llj Vol. 11, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 161 urogenital sinus, and male-type external genitalia in the female fetus is limited to a sharply defined period of fetal life. Once the future differentiation or regression of these structures has been determined, it is no longer possible to modify their development. Treatment after this stage has an appreciable effect only on the growth of the clitoris. This concept is also supported by the masculinizing effects on the genital tract of the monkey of testosterone injected at various fetal ages. In the monkey, large doses of testosterone propionate administered too early in gestation (twenty-fifth to thirtieth day), before completion of the initial phase of organogenesis of the genital tract, did not lead to female pseudohermaphrodism in the fetus. 26 In general, the masculinizing activity of an androgen in the female fetus varies directly with its androgenic pote;.cy in the adult animal. However, this relationship is not an invariable one. Jost 8 has shown that methylandrostenediol dipropionate, a weak androgen in the adult rat, has a potent masculinizing action on the fetus. In addition to the factors of dosage, androgenic potency, fetal age, and species differences in end-organ receptivity, genetic sex may also influence the growth capacity of the sex primordia. Bums has demonstrated a sex difference in the capacity for growth of male derivatives of the urogenital sinus in androgen-treated male and female pouch young of the opossum.2'f The evidence bearing on this point in placental mammals is inconclusive. In this discussion, consideration of the action of androgens on the fetus has been limited to their influence on the sex primordia. Two other effects deserve mention. The administration of androgens, including 17-ethvnyltestosterone, to lower placental mammals during pregnancy results in a high incidence of abortion or resorption of the fetal organism This injurious effect is less evident, although demonstrable, in the rhesus monkey Josfl 29 has described a deleterious effect on the growth of the rat fetus of certain androgens injected late in pregnancy. There is evidence that the latter is an indirect, rather than direct, effect on the fetus; castration of the pregnant rat and the injection of progesterone in addition to androgen prevents the inhibition of fehil growth. 80 EFFECT OF- ANDROGENS ON THE HUMAN FETUS Recently, Salhanick8 1 showed that labeled testosterone administered to the mother during the twelfth week of pregnancy is rapidly transferred

6 162 GRUMBACH & DUCHARME Fertility & Sterility across the placenta. There is indirect evidence that certain analogues of testosterone are also readily transmitted from the mother to the fetus. The effects of androgens on fetal sex differentiation in man are comparable to those obtained experimentally in other placental mammals. The androgen-induced forms of human female pseudohermaphrodism clearly demonstrate that exposure of the female fetus to hormones with androgenic activity, whether of fetal or maternal origin, is capable of modifying the development of the urogenital sinus and the external genitalia along masculine linesy These substances, however, do not induce differentiation of derivatives of the Wolffian ducts. By far, the commonest cause of fetal masculinization is congenital virilizing adrenal hyperplasia in females; the fetus, in this instance, is the source of androgen. In this disorder, a genetically-determined enzymatic defect in the biosynthesis of hydrocortisone, transmitted as an autosomal recessive trait, results in excessive secretion of androgenic steriods by the fetal adrenal gland. Masculinization of the urogenital sinus and/ or the external genitalia in females also has been caused by androgens transmitted from the mother during pregnancy. In two published instances, the source of androgenic steroids was a maternal arrhenoblastoma More commonly, the maternal source has been steroids with androgenic activity administered to the mother, and the virilization of the fetus has been iatrogenically produced. These androgenic preparations include testosterone and its analogues and the semisynthetic oral progestins: 17 a-ethynyltestosterone ( Lutocylol, Pranone), 19-nor-17 a-ethynyltestosterone ( N orlutin), and norethynodrel (En ovid). Less clearly characterized is the association with progesterone 8 and diethylstilbestrop 4 In experimental female pseudohermaphrodism, the age of the fetus is a crucial factor in determining the effect of androgen on the sex primordia. Instances of iatrogenic masculinization of the female fetus facilitate critical analysis of the role of the age factor in the pathogenesis of the genital anomalies in man. Sex differentiation takes place in three phases, involving successively ( 1) the gonad, ( 2) the genital ducts, and ( 3) the urogenital sinus and the external genitalia. The length of the indifferent period of development of each of these structures varies; an outline and diagrammatic scheme of the sequence of differentiation of the lower genital tract, constructed from sev- ii

7 Vol. 11, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 163 TABLE 2. Differentiation of the External Genitalia Fetal age (weeks) Differentiation Indifferent stage Appearance of genital tubercle Appearance of urethral folds, urethral groove, and genital swellings Male Fetal penis Fusion of urethral folds in progress; Urogenital ostium extends to coronary sulcus Rounded genital swellings located toward anus Perineal raphe Urinary meatus at tip Differentiated stage Female Phallus bent caudally Urethral groove open wide Elongated genital swellings flank base of phallus Posterior commissure formed by fusion of caudal ends of genital swellings Clearly female in character Definitive female urethra Canalization of female genital tract complete eral sources,35-40 is shown in Table 2 and Figs. 1 and 2. The estimate of fetal age, based on crown-to-mmp length, is derived from the tables of Hamilton et al. 41 and of Witschi 42 and, in view of the considerable variation in fetal length at a specific developmental stage, should be regarded only as an imprecise estimate of actual age. In addition, little is known about the range of normal variation in fetal age during which a specific developmental change may occur. Exposure of the female human fetus to hormones with androgenic activity during a critical phase of sex differentiation may inhibit descent of the "uterovaginal complex" and female development of the urogenital sinus, and may induce differentiation of a prostate gland and varying degrees of masculinization of the external genitalia (Fig. 2). The latter include fusion of the urethral folds (which, in some instances, may extend the orifice of the urogenital sinus onto the shaft of the phallus-rarely to the tip), posterior descent of the genital swellings to produce a scrotum or scrotumlike structure, and the development of a penislike phallus. Lesser degrees of masculinization result in the development of external genitalia which are more ambiguous in appearance or in which, except for an enlarged phallus, the

8 164 GRUMBACH & DUCHARME Fertility & Sterility INDIFFERENT Genital tubercle Phallus 25mm-8th wk 15mm-7th wk MALE FEMALE Scrot IOOmm US-16th wk Vestibule Fig. I. A schematic diagram of the differentiation of the external genitalia (adapted from Wilson,:n Glenister,as and Spaulding-'~9). external genitalia are female in character ( Fig. 2). In sum, androgens simulate the action of the fetal testicular secretion on the urogenital sinus and the external genitalia, but do not appreciably effect differentiation of. the genital ducts or, insofar as is known, the gonad.

9 ANDROGENS AND FETAL SEXUAL DEVELOPMENT Vol. 11, No.2, 1960 A. 50mm h wk 165 B. Ulero-vogmol 70mm Muller o~mple~ luberc~e-~ Clitoris Fig. 2. Upper diagrams show the sequence of differentiation of the female accessory sex sb uctures. Note the gradual descent of the uterovaginal "complex." Canalization of the vagina and uterus and definitive differentiation of the vestibule are not complete before the fifth month (adapted from Koff3 5 ). Lower diagrams schematically illustrate variations in the degree of fetal masculinization of the urogenital sinus and external genitalia in androgen-induced female pseudohermaphrodism. IATROGENIC FETAL MASCULINIZATION The inadvertent occurrence of partial masculinization of the female fetus, associated with treatment of pregnant women with androgens, provides a unique opportunity to examine various factors which affect the action of these steroids on the primitive genital tract, and to obtain further indirect evidence of the placental transfer of these substances. Thirteen cases of masculinization of the female fetus following treatment of the mother with hormone have been recorded in previous reports (Table 3). We have added a case (see Appendix and Fig. 3) that was studied at Babies Hospital. The data of the 10 patients43-52 in whom significant fusion of the labioscrotal folds was present are summarized in Table 3, Cases In all but Case 3, treatment of the mother had been instituted before what was esti-

10 166 GRUMBACH & DUCHARME Fertility & Sterility TABLE 3. Data on 14 Cases of Masculinization of the Female ~aternalhorntonaltherapy Dosage Total Age of schedule Time in dose Source Case no. patient Preparation and route pregnancy (Gm.) Hayles & 7 months Methy!testosterone 10 mg. b.i.d. ir- 7th wk. to term 1.5 Nolan4-8 regularly; buccal Grunwaldt & 2 6 months Methy!testosterone 10 mg. t.i.d., p.o. 8th to 15th wk Bates44 (51 days) Testosterone pro- 100 mg. x 2, I.M. 8th wk pionate Carpentier45 Stillborn 19-Nor-methyltes- 2.5 mg. q.i.d., p.o. 12th to 2:lrd wk. Ca (1040 Gm.) tosterone Progesterone 50 mg. x 8, l.m. 23rd wk Gold& 4 Newborn Methyltestosterone 30 mg./<lay, p.o. 7th to 14th wk Michael46 Premarin 3.75 mg./day, p.o. 7th to 14th wk Nellhaus days Methy!testosterone 10 mg. b.i.d., p.o. 3rd to 17th wk. 2.0 Ethinylestradiol 0.04 mg. b.i.d. 3rd to 17th wk Foxworthy weeks Methyltestosterone 10 mg./nay, p.o. 7th wk. to 6 mo. Ca. 1.0 Premarin 125 mg./day, p.o. 7th wk. to 6 mo. Ca Progesterone 25 mg./day, I.M. 4th to 7th wk. Ca Bongiovanni49 7 Child Methylandro- 50 mg./wk., I.M. loth wk. to 8th mo. stenediol Moncrieff years Methyltestosterone 3 mg. b.i.d., p.o. } For 1 mo. prior to Ethinylestradiol 0.01 mg. b.i.d.. p.o. preg. diagnosis Methyltestosterone } Ca mg. t.i.d., p.o. }F t 2 Ethinylestradiol 0.01 mg. t.i.d., p.o. or nex mo. Progesterone 50 mg. implant 3rd and 4th mo Nilsson & 9 Newborn Methylandrostene- 25 mg./day, Parts of 3rd and? Soderhjelm51 ldiol dipropionate sublingual 4th mo. Black & 10 6 weeks Methyltestosterone 5 mg. b.i.d., p.o. 2nd to 5th mo. Ca Bentley52 Zander& 11 Newborn Methy landro- 100 mg. b.i.w., 22nd to 38th wk. 3.2 MullerG3 stenediol I.M. Hoffman 12 Newborn Testosterone 65 mg. X 13, l.m. 4th to 9th mo. 845 mg. et al.5g enanthate Estradiol valerate 4 mg. x 13, I.M. 4th to 9th mo. 52 mg. Methyltestosterone 28 mg./wk., p.o. 9th mo. 112 mg. Moncriefl' weeks Methyltestosterone 10 mg. t.i.d., p.o. Ca. 3rd mo. for weeks 10 mg. t.i.d., p.o. Ca. 6th mo. for 6 weeks This report (see months Methyltestosterone 10 mg./day, Appendix) buccal 14th wk. to term 1.82 Testosterone pro.. 25 mg., I.M. 14th, 27th, 31st wk. 115 mg. pion ate 40 mg., I.M. 35th wk.

11 Vol. 11. No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 167 Fetus Following Treatment of the Mother with Androgens External genitalia of female offspting Urinary ClitOTal Labio- 17-ketosenlarge- scrotal tetoids ment fusion (mg./24 hr.) Se.~ chromatin pattern Signs of virilization in mother Indication for tteatment during pregnancy Not detected 1.5 em. in Complete; phallic 0.3 length urethra5 b Positive Positive Deepening of voice, hirsutism, elitoral enlargement Deepening of voice Alopecia Nausea and vomiting em. in Complete; phal length lie urethra Positive Positive Deepening of voice, seborrhea, loss of scalp hair None Previous abortions Osteoporosis 1.0 em. in length Positive Deepening of voice Previous abortions Normal Positive Previous abortion Normal Positive Positive Weakness and lowb.p. Previous abortions ~ None 0.14~ X 0.6 None 1.7 em. Positive Positive None Deepening of voice, hirsutism Deepening of voice Deepening of voice Vomiting Previous abortions; prescribed in error Sarcoma of breast Pruritus + None Positive Vaginal bleeding 4.0 X 1.5 None em. Positive None Breast engorgement 7 months postmammoplasty a Laparotomy: Normal uterus, tubes and ovaries. "Urethrogram: Urogenital sinus and vagina. Surgical repair: Vagina visualized at surgery at 8 months of age. d Autopsy: Female internal genitalia.

12 4 Fig. 3. The appearance of the external genitalia in Case 14 (see Appendix). Note enlargement of the clitoris without fusion of the labioscrotal folds. Fig. 4. An intermediate degree of masculinization of the external genitalia in a 2-week-old female pseudohermaphrodite with congenital virilizing adrenal hyperplasia. The enlarged clitoris, bound in chordee, overlies the funnel-shaped orifice of the urogenital sinus. Fig. 5. Enlargement of the clitoris without fusion of the labioscrotal folds in a 4-year-old female with virilizing adrenal hyperplasia. Hypertrophy of the clitoris was noted at birth. Separate vaginal and urethral orifices were identified by inspection. Note, also, the pubic hair. 5

13 Vol. 11, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 169 mated to he the twelfth week of pregnancy; in Case 3 treatment was started during the twelfth to thirteenth week. Cases 2 and 4 showed complete fusion of the labioscrotal folds and a penile urethra. In both instances, substantial doses of androgen had been given during the third month of pregnancy. In general, the degree of labioscrotal fusion is directly related to the amount of hormone given to the mother between the eighth and thirteenth weeks of pregnancy. It is of interest that the size of the clitoris did not always correlate with the extent of labioscrotal fusion. Cases in which treatment had been terminated before the middle of the second trimester had a relatively small phallus as judged from photographs of the external genitalia. On the other hand, female infants with congenital adrenal hyperplasia examined during the neonatal period exhibit phallic enlargement which is proportional to, or greater than, the degree of labioscrotal fusion; occasionally the latter may even be minimal or absent. It seems probable that the relatively small size of the phallus in the androgen-treated cases is related to the limited period of treatment as well as to the dose and potency of androgen. The mothers of 4 of the 10 patients (Cases 4, 5, 6, and 8) had also received varying amounts of estrogen, and the mothers of 3 patients ( Cases 3, 6, and 8) had been given progesterone. The effects of androgen on the fetus in these cases did not seem to differ from those in which androgen alone was administered. The data in 4 cases in which the patient showed clitoral enlargement without significant labioscrotal fusion are shown in Table 3, Cases In 3 of the cases, androgen was administered to the mother after the twelfth week of pregnancy. In Moncrieff's 50 case ( 13) reported in a brief preliminary note, it is uncertain when in the third month of pregnancy the first course of treatment with methyltestosterone was started. Although substantial amounts of androgen had been given to the mothers of these 4 patients, fusion of the labioscrotal folds was not observed (Fig. 3). It seems probable that differentiation of these structures had been determined prior to the administration of androgen. About the twelfth week of gestation there is a definite sex difference in the appearance of the external genitalia (Table 2, Fig. 1), However, differentiation of the lower female genital tract is not yet complete (Fig. 2) 35 and it is of interest that development of the vagina in these cases was not modified. Hence, the action of androgen on the genital structures of the female fetus after the end of the third month of gestation

14 170 GRUMBACH & DUCHARME Fertility & Sterility is comparable to the alterations produced by postnatal virilism in females; namely, enlargement of the clitoris and of the labia majora. In 2 cases a gradual decrease in size of the enlarged clitoris was observed The androgenic activity in the adult of two of the testosterone analogues- 19-nor-17 a-methyltestosterone and 17 a-methyl-~ 5 -androstene-5,8, 17,8-diol -is relatively low. On the other hand, the data contained in Table 3 indicate that these substances may have a potent androgenic effect on the fetus. This discrepancy in action could be attributable to differences in placental transfer and in the sensitivity of fetal and adult end-organs, but it is also likely that a difference in the fetal metabolism, especially the rate of disposal of these preparations, plays an important role.8 The occurrence or absence of signs of virilization in the mother was specifically mentioned in ten reports. In seven instances androgenic effects were noted in the mother; usually these were mild. In three instances the mother did not exhibit evidence of virilization. In general, there was a striking disparity between the signs of virilization in the mother and those in the female fetus. J ost 3 29 found that the administration of certain androgens to pregnant " rats was associated with a reduction in the weight of the fetus. Black and Bentley 52 noted that the birth weight of their patient and that of 2 other cases was low in relation to the length of gestation. However, in 5 other cases (2, 4, 5, 9, and 14) the birth weight was normal. Treatment with male hormone during pregnancy does not invariabl) result in masculinization of the female fetus. V andekerckhove55 has described a pregnant woman with carcinoma of the breast who was given a total of 6850 mg. of methylandrostenediol sublingually. Treatment was begun during the first month of pregnancy, and neither the patient nor her female offspring exhibited signs of virilization. The "indications" for which the mothers were given male hormone therapy during pregnancy are listed in Table 3. According to the experimental and clinical data presented, there is an evident danger to the female fetus from exposure to excessive amounts of hormones with androgenic activity. In view of the lack of clearly defined rationale or evidence.of the therapeutic value, treatment with male hormone is contraindicated during pregnancy. THE ANDROGENIC EFFECTS OF CERTAIN ORAL PROGESTINS The occurrence of partial masculinization of the external genitalia in female infants whose mothers were given oral 17 -ethynyltestosterone ( Lutocylol, Pranone) or parenteral progesterone has been reported by Wilkins

15 Vol. 11, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 171 et au In 15 of these cases the mother had been treated with 17 -ethynyltestosterone and, in 3 cases, with intramuscular injections of progesterone. It was suggested that the fetal masculinization was caused by an abnormal metabolism of these steroids by the mother, or by an abnormality in placental transfer. We have studied eighteen instances of partial masculinization of the female fetus that were associated with oral progestin therapy during pregnancy. Our experience is reported elsewhere 8 and will be summarized briefly. In 9 cases the mother had received 19-nor-17 ethynyltestosterone ( N orlutin); in 1 case, norethynodrel ( Enovid); and in 8 cases, 17-ethynyltestosterone. Norlutin had an androgenic action on the fetus comparable to that of methyltestosterone, while 17-ethynyltestosterone was appreciably less potent. Labioscrotal fusion was exhibited only in those instances in which the oral progestin had been administered prior to the thirteenth week of gestation. It must be emphasized that testosterone and a number of its methyl, ethyl, ethynyl, and 19-nor analogues exhibit a progestational action on the endometrium in animal assays and in man In some instances the progestational potency greatly exceeds that of injected progesterone. 56 However, none of these steroids is capable of maintaining pregnancy in the castrated pregnant rat, 57 and they may even have a deleterious effect on pregnancy in the intact animal, As far back as 1942, Courrier and JoseH, 38 demonstrated that 17a-ethynyltestosterone could produce masculinization of the female rabbit fetus and cautioned about its use in pregnant women. Data of this kind in experimental animals are not available for the newer "oral progestins," although experiments in castrated adult animals have shown these substances to be weakly androgenic in the mature animap 9 Nonetheless, clinical evidence 8 strongly suggests that these testosterone analogues with progestational activity may have a potent, direct androgenic action on the human female fetus-an activity which correlates poorly with that obtained by classic methods of androgen bioassay. The discrepancy between the androgenic properties of these oral progestins in the adult and in the fetus could be attributed to differences in the responsiveness of receptors/ 0 27 to delayed fetal disposal of steroid transferred across the placenta, or to a combination of the two. Recently, we reviewed evidence 8 which supports the contention that the androgenicity of certain oral progestins in the fetus is related to differences in fetal metabolism-particularly the rate of disposal of these steroids.

16 172 GRUMBACH & DUCHARME Fertility & Sterility In the light of these observations, it is our view that oral progestins which exhibit androgenic activity in the female fetus should not be used in the treatment of pregnant women. 8 Although available data are insufficient to permit even a crude approximation of the incidence of congenital masculinization of female fetuses following administration of these preparations during pregnancy, our experience with Norlutin suggests that the risk may be appreciable. The importance of dosage, duration of therapy, and the time in pregnancy during which the fetus is exposed in determining the extent and degree of masculinization has already been emphasized, as has individual variation in susceptibility to the effects of androgen. It should also be mentioned that unless abnormalities of the external genitalia are specilically sought in female infants, they may readily be overlooked. On the other hand, there is inconclusive clinical evidence 8 to implicate progesterone7 60 itself in fetal masculinization. It is true that progesterone, when administered in large doses, has a weak androgenic effect on certain receptors of castrate male animals However, an androgenic action on the female fetus has not been demonstrated in pregnant experimental placental mammals. It is important to distinguish the fetal masculinizing properties of testosterone analogues with oral progestational activity from those rare instances of congenital masculinization of the female fetus in which a relationship to treatment during pregnancy with progesterone7 60 or its analogues, or diethylstilbestrol, 34 has been suspected but not established. 8 THE FETAl ADRENAl GlAND AS A SOURCE OF ANDROGEN: CONGENITAl VIRiliZING ADRENAl HYPERPlASIA In females, virilizing adrenal hyperplasia (beginning in fetal life) causes, through excessive secretion of androgenic steriods by the abnormal fetal adrenal gland, masculinization of the urogenital sinus and/ or the external genitalia (Fig. 2). In the majority of instances there is an intermediate degree of masculinization 63 of the lower genital tract (Fig. 4), but in some patients there is present at birth only clitoral enlargement (Fig. 5) or, more rarely, a penile urethra (Fig. 6). The factors responsible for the variations in the genital anomalies in this form of androgen-induced female pseudohermaphrodism are poorly understood. It seems probable that gradations in the enzymatic defect in hydrocortisone synthesis, and the time in pregnancy when this defect becomes manifest are important considerations. However, there is evidence that other factors peculiar to the fetal environment may also be of importance. The

17 Vol. 11, No.2, ANDROGENS AND FETAL SEXUAL DEVELOPMENT 8 A. c Fig. 6. (A) A 45-month-old female pseudohermaphrodite with congenital adrenal hyperplasia and a penile urethra who has been reared as a male. Height is 112 em. ( +2.5 S.D.); bone age, 8 years; urinary 17-ketosteroids: 11:5 to 13.8 mg. per day; and pregnanetriol, 3 to 4 mg. per day. The sex chromatin pattern was positive. (B) The appearance of the external genitalia. (C) Urethrogram shows the urogenital sinus and a distended vagina. Some of the contrast medium also entered the bladder. adrenal primordium first appears in human embryos 64 during the sixth week ( 8- to 10-mm. stage). Of interest is the fact that it reaches its maximal relative size in the third or fourth fetal month during the critical period of differentiation of the accessory sex structures. Normally the fetal adrenal does not seem to play a significant role in sex differentiation. The relationship between time of exposure of the fetus to androgen and the extent of masculinization of the external genitalia and urogenital sinus in iatrogenic female pseudohermaphrodism has a bearing on the pathogenesis of congenital adrenal hyperplasia. This association suggests that, in the latter dis-

18 174 GRUMBACH & DUCHARME Fertility & Sterility order, the abnormal fetal adrenal cortex is secreting appreciable amounts of androgenic steriods during the third month of gestation in affected females who have fusion of the labioscrotal folds and a urogenital sinus. Solomon et al. 65 have demonstrated that homogenates of human fetal adrenals, * when incubated with progesterone, can synthesize androstenedione and 17a-hydroxyprogesterone. It is not known whether in early fetal life increased circulating fetal ACTH provides the stimulus for excessive secretion of androgen by the abnormal fetal adrenal (as is the case postnatally) or whether additional factors are involved. The available data are insufficient to resolve this problem. VIRILIZING OVARIAN TUMORS IN PREGNANCY AS A MATERNAl SOURCE OF ANDROGEN Androgenic hormones transmitted to the female fetus from maternal virilizing ovarian tumors have caused partial masculinization of the urogenital sinus and the external genitalia.u Two female pseudohermaphrodites have been described whose mothers had an anhenoblastoma during pregnancy. Brentnall's patient, 33 whose mother had noted signs of virilism by the fourth month of pregnancy, was a female with labioscrotal fusion and enlargement of the clitoris. In the mother of Felicissimo's and de Abreu's case, 32 recurrence of an arrhenoblastoma in the fifth month of pregnancy produced signs of virilization; the female infant was born with an enlarged phallus, but without labioscrotal fusion. In 3 cases of virilizing ovarian tumors during pregnancy, the female infants were apparently normal. In 1 case, 66 an ovarian mass detected in the fifth month of pregnancy (at the first prenatal examination) was removed during the eighth month, following rapid progression of virilization. The patient of Alexander and Beresford 67 first detected virilizing signs at about the fifteenth week of pregnancy. Why the female offspring in the latter two instances did not exhibit clitoral enlargement is not clear; it is possible that a minor degree of hypertrophy may have been overlooked. A decrease in secretion of androgen by the ovarian tumor prior to and during pregnancy may have occurred in the patient reported by Eckman. 68 FEMAlE PSEUDOHERMAPHRODISM OF UNDETERMINED ETIOlOGY Apart from the rare instances of nonadrenal female pseudohermaphrodism * Two thirds of the glands were obtained from fetuses 14 to 18 weeks of age, and the remainder from 10- to 22-week fetuses.

19 Vol. 11, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 175 associated with renal or cloacal anomalies 7 11 in which an unknown teratogenic factor appears to be involved, or those instances in which the female fetus has been exposed to a known source of androgen, there is an additional rare group of female pseudohermaphrodites of obscure etiology. The configuration of the external genitalia in these latter patients is identical to that seen in the androgen-induced forms. In 3 such cases studied at Babies Hospital, careful inquiry into the maternal history during gestation was unrevealing; the patients did not excrete abnormal amounts of urinary steroids, and the adrenal glands and ovaries were normal at exploratory laparotomy. In the case of these patients we can only suspect that the fetus had been subjected to excess androgen, perhaps temporarily, from some maternal or fetal source. DIAGNOSIS AND MANAGEMENT OF FEMALE PSEUDOHERMAPHRODISM Female pseudohermaphrodism must be distinguished from other forms of intersexuality in which there is bilateral cryptorchidism. The configuration of the external genitalia is not a distinctive characteristic. The history may reveal other siblings affected with congenital virilizing adrenal hyperplasia, signs of progressive virilization, or evidence of dehydration, vomiting, and collapse suggestive of an Addisonianlike electrolyte disorder. The mother and the obstetrician should be queried concerning hormones administered during pregnancy and the occurrence of signs of virilization. The detection of chromatin-positive nuclei quickly limits the diagnostic possibilities to some form of female pseudohermaphrodism or to true hermaphrodism with undescended gonadsy In the latter, the sex chromatin pattern is more frequently positive than negativey Twenty-four-hour specimens of urine should be examined for total 17-ketosteroids and, when possible, for pregnanetriol and 17 -ketogenic steroids. In virilizing adrenal hyperplasia these steroids are excreted in increased amounts, but not always in the first weeks of life. Serum electrolyte concentrations should be measured in any infant in whom adrenal hyperplasia is suspected. It is sometimes advisable to inject a radiopaque contrast medium into the single perineal orifice to outline the urogenital sinus under fluoroscopic examination (Fig. 6) when a separate nrethral and vaginal orifice cannot be identified by inspection. An intravenous pyelogram is of value for the detection of anomalies of the urinary tract in the nonandrogen-induced fonns of female pseudohermaphrodism.

20 176 GRUMBACH & DUCHARME Fertility & Sterility The diagnosis of congenital adrenal hyperplasia is readily established by clinical and laboratory studies, and surgical exploration of the pelvis is superfluous. In addition, we no longer subject to laparotomy patients who have chromatin-positive nuclei and normal values for urinary steroids, and whose mothers were treated during pregnancy with hormones implicated as potential fetal masculinizing agents. In the other rare forms of female pseudohermaphrodism, laparotomy and bilateral gonadal biopsy are necessary for a definitive diagnosis. It is of great importance, for infants with abnormal external genitalia, that diagnostic studies that include determination of the sex chromatin pattern and excretion of urinary 17 -ketosteroids not be deferred until a late age. Female pseudohermaphrodism is the most satisfactory form of ambisexual development to treat when it is recognized early. In congenital virilizing adrenal hyperplasia the administration of corticosteroid therapy prevents or arrests virilization and acceleration of osseous development. The genital defect in female pseudohennaphrodites is readily corrected by appropriate surgical procedures. With modern methods of treatment, the psychological, physical, and social impediments which may result from the abnormality can be avoided or minimized. In addition, the potential for fertility is good. Elsewhere in this journal Dr. Howard Jones discusses in detail the procedure for plastic repair of the genital defect; one additional point deserves mention. Dr. John Lattimer, of the Department of Urology, has developed a procedure for clitoral recession in patients without adrenal hyperplasia in whom clitoral enlargement presents a problem. This operation, which avoids amputation of the clitoris, has been especially useful in patients with partial masculinization of the external genitalia that resulted from the administration of steroids with androgenic activity to the mother during pregnancy. In these patients, abnormal growth of the clitoris does not occur after birth; none has required a clitorectomy. Case History APPENDIX The patient's mother, now para 6, gravida 5, had had pregnancies in 1948 and 1950 each of which resulted in the birth of two norn1al boys. In July 1950, the mother had 5 "cysts" removed from both breasts and a mammoplasty performed for cosmetic reasons. The last menstrual period preceding the third

21 Vol. II, No. 2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT 177 pregnancy was on November 14, Because of engorged and painful breasts, her physician prescribed methyltestosterone ( Ciba), 10 mg. daily, sublingually. Treatment was begun on March 8, 1951, and continued at the same dosage until the day of delivery, August 16, In addition, the mother was given during this pregnancy four injections of testosterone propionate (Perandren, Ciba) intramuscularly: 25 mg. on March 8, June 7, and July 5, and 40 mg. on August 2. Pregnancy was otherwise uneventful. The mother and obstetrician did not note any signs of virilization, and no other hormones were administered. The patient weighed 3860 gm. at birth. The mother was not aware that the patient's external genitalia were abnormal until shortly before the child's admission to Babies Hospital in July 1955 at 46 months of age. The parents first noted and became concerned about the enlarged clitoris when the patient complained of pain in the perineal region; apparently the result of a minor irritation of the skin. Neither parent recalled having examined closely the external genitalia prior to this time. Since early infancy the patient had been seen only occasionally by a physician for minor illnesses. Somatic growth and mental development were unremarkable. No signs of progressive virilization had been observed. When examined at admission, height ( 105 em.), weight ( 17.0 kg.), and dentition were normal. No secondary sexual characteristics, deepening of the voice, or excessive muscular development were present. The only abnormality detected was a significantly enlarged clitoris. There was a separate vaginal and urethral orifice and no fusion of the labioscrotal folds. A midline mass consistent with a small uterus was palpated on rectal examination. The remainder of the physical examination showed no abnormalcy. When the patient was re-examined at 7~ years of age, the clitoris was still conspicuously enlarged (Fig. 3), measuring 4.0 X 1.5 em., and contained a firm corpus spongiosum. The glans, which was prominent, was partly covered by preputial skin. The labia minora and the vaginal mucosa were infantile, and the uterus was of prepubertal size. No signs of virilization or sexual precocity were detected. A high percentage of chromatin-positive nuclei were seen in oral mucosal smears. Determinations of urinary 17 -ketosteroids obtained at 46 months and 7~ years were normal (range 2.3 to 3.2 mg./ 24 hours). Osseous development was consistent with chronologie age. REFERENCES 1. EMMENS, C. W., and PARKES, A. S. The effect of route of administration on the

22 178 GRUMBACH & DUCHARME Fertility & Sterility multiple activities of testosterone and methyltestosterone in different species. f. Endocrinol. 1:323, DoRFMAN, R. I., and SHIPLEY, R. A. Androgens. Biochemistry, Physiology, and Clinical Significance. New York, John Wiley & Sons, 1nc., 1956, Chapter JosT, A. Croissance des cmbryons chez des femelles de rat injectees d'androstimediol, et de methylandrostimediol, ou castrees. Ann. endocrinol. (Paris)16:283, KLEIN, M., and PARKES, A. S. Progesterone-like action of testosterone and certain related compounds. Proc. Roy. Soc. (London) ser. B 121:574, HisAw, F. L. Androgens and experimental menstruation in the monkey (Macaca mulatta). Endocrinology 33:39, MASTERS, \V. H., and MAGALLON, D. T. Androgen administration in the postmenopausal woman. ]. Clin. Endocrinol. 10:348, WILKINS, L., JoNES, H. \V., JR., HoLMAN, G. H., and STEMPFEL, R. S., JR. Masculinization of the female fetus associated with administration of oral and intramuscular progestins during gestation: non-adrenal female pseudohermaphrodism. ]. Clin. Endocrinol. 18:559, GRUMBACH, i\l M., DucHARME, J. R., and MoLOSHOK, R. E. On the fetal masculinizing action of certain oral progestins. ]. Clin. Endocrinol. 19:1369, JosT, A. Problems of fetal endocrinology: the gonadal and hypophyseal hormones. Recent Frog. Hormone Research 8:379, JosT, A. La fonction endocrine du testicule foetal. In La Fonction Endocrine du Testicule. Paris, Masson et cie, 1957, pp GRUMBACH, M. M., and BARR, M. L. Cytologic tests of chromosomal sex in relation to sexual anomalies in man. Recent Frog. in Hormone Research 14:255, BuRNS, R. Hormones and the differentiation of sex, In Survey of Biological Progress, AvERY, G. S., JR. et al. (eds.) New York, Academic Press, Inc., Vol. 1, pp GALLIEN, L. The action of sex hormones on the development of sex in amphibia. Mem. Soc. Endocrinol. (Cambridge) No. 4:188, JosT, A. Modalities in the action of gonadal and gonad-stimulating hormones in the foetus. Mem. Soc. Endocrinol. (Cambridge) No. 4:237, MooRE, C. R. Embryonic Sex Hormones and Sexual Differentiation. A monograph in American Lectures in Endocrinology. Willard 0. Thompson ( ed.). Springfield, Charles C Thomas, WELLS, L. J. Hormones and sexual differentiation in placental mammals. Arch. anat. miscrosc. morph. exp. 39:499, BRUNER, J. A., and vvitschi, E. Testosterone-induced modifications of sex development in female hamsters. Am. ]. Anat. 79:293, WHITE, M. Effects of hormones on embryonic sex differentiation in the golden hamster. II. Estrogenic effects in treated males; androgenic and estrogenic effects in treated females. ]. Exp. Zool. 110:153, GREENE, R. R., BunRILL, M. W., and IvY, A. C. Experimental intersexuality: the effect of antenatal androgens on sexual development of female rats. Am. ]. Anat. 65:415, BuRRILL, M. W., and GREENE, R. R. Experimental intersexuality: correlation between treatment and degree of masculinization of genetic female rats. Am. ]. Physiol. 126: p. 452, MooRE, C. R. Prostate gland induction in the female opossum by hormones and the capacity of the gland for development. Am. ]. Anat. 76:1, JosT, A. The age factor in some prenatal endocrine events. Ciba Foundation Coli. Ageing 2:18, 1956.

23 Vol. 11, No.2, 1960 ANDROGENS AND FETAL SEXUAL DEVELOPMENT BuRRILL, M. W., GREENE, R. R., and IvY, A. C. Experimental intersexuality: lateral asymmetry in the rat intersex. Anat. Rec. 76:173, VANWAGENEN, G., and HAMILTON, J. B. The experimental production of pseudohermaphroditism in the monkey, In Essays in Biology. Berkeley, Univ. of California Press, 1943, pp DANTCHAKOFF, v. Sur les mecanismes differentiels dans la realisation des sexes chez les vertebres superieurs (Singe Macacus rhesus). Bull. biol. France et Belg. 84:311, WELLS, L. J., and VANWAGENEN, G. Androgen-induced female pseudohermaphroditism in the monkey ( Macaca mulatta): anatomy of the reproductive organs. Embryol. 35:93, BURNS, R. K. Hormones versus constitutional factors in the growth of embryonic sex primordia in the opossum. Am. ]. Anat. 98:35, JosT, A. Recherches sur la differenciation sexuelle de l'embryon de lapin. 2. Action des androgt'mes de synthese sur l'histogenese genitale. Arch. anat. microsc. morph. exp. 36:242, JosT, A. Action sur la croissance des embryons de divers androgenes injectes ala rate gestante. Ann. endocrinol. (Paris) 17:118, MAROIS, M. Androstenediol, ovaire et croissance foetale. C. R. Soc. Biol. 151: 646, SALHANICK, H. A. Personal communication. 32. FELICISSIMO, PAULA XAVIER, J., and DE ABREU JuNQUEIRA, M. Sobre un caso de arrhenoblastoma de ovario e gravidez topica simultanea. Virilisac;ao de gestante e do feto feminino. Rev. (!.ynec. e obst. (Rio de Janeiro) 32 ( 1) :356, BRENTNALL, C. P. Case of arrhenoblastoma complicating pregnancy. ]. Obst. & Gynaec. Brit. Emp. 52:235, BoNGIOVANNI, A.M., m GEORGE, A.M., and GRuMBACH, M. M. Masculinization of the female infant associated with estrogenic therapy alone during gestation: four cases. ]. Clin. Endocrinol. 19:1004, KoFF, A. K. Development of the vagina in the human fetus. Embryol. 24:59, HuNTER, R. H. Observations on development of the human female genital tract. Embryol. 22:91, WILSON, K. M. Correlation of external genitalia and sex glands in the human embryo. Embryol. 18:23, GLENISTER, T. W. The origin and fate of the urethral plate in man. ]. Anat. (London) 88:413, SPAULDING, M. H. The development of the external genitalia in the human embryo. Embryol. 13:67, WITSCm, E. Sex chromatin and sex differentiation in human embryos. Science 126:1288, HAMILTON, W. J., BoYD, J. D., and MossMAN, H. W. Human Embryology. Prenatal Development of Form and Function. Cambridge, W. Heffer and Sons, 2nd ed., 1952, p WITscm, E. Development of Vertebrates. Philadelphia, W. B. Saunders, 1956, pp HAYLES, A. B., and NoLAN, R. B. Female pseudohermaphroditism: Report of case in an infant born of a mother receiving methyltestosterone during pregnancy. Proc. Staff Meet. Mayo Clinic 32:41, Gmr~ALDT, E., and BATES, T. Nonadrenal female pseudohermaphrodism after administration of testosterone to mother during pregnancy. Report of case. Pediatrics 20:503, 1957.