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THE MINIMUM QUANTITY OF REQUIRED TO INDUCE ATYPICAL EPITHELIAL GROWTH OF THE UTERINE MUCOSA IN THE GUINEA-PIG

ALEXANDER LIPSCHUTZ, M.D., LUIS VARGAS, JUN., M.D.. ARMANDO JEDLICKY, M.D., AND PEDRO BELLOLIO, M.D. (From the Department of Experimental , National Health Service, Republic of Chile, Santiago)

It has been shown by many workers that a cystic glandular of the uterine mucosa can be induced in various species by prolonged admin- istration of follicular hormones. There may occur, also, a metaplasia of the of the , which becomes stratified in certain areas, while the glands may be transformed into solid epithelial cords. Under simi- lar experimental conditions metaplastic changes may occur also in the uterine (for literature see Gardner, 1, and Loeb, 2). These observations are of special importance since the metaplastic changes, which sometimes assume a precancerous aspect, may persist even when administration of is suspended. In one case atypical growth of the uterine epithelium of precan- cerous aspect was observed in a castrated guinea-pig which had received sub- cutaneous injections of the monobenzoic of for a period of eight months, though the injections were suspended four months before the animal was sacrificed (Lipschutz, Iglesias and Vargas, 3). Allen and his associates (4) were successful in transplanting a cancerous cervical tumor occurring in a mouse treated with estrogens for ten months. It may be objected that in all these experiments an artificial condition is created and that no conclusions can be based upon them as to spontaneous tumorigenesis by endogenous estrogens, i.e. by estrogens secreted by the in situ. On the other hand, it was shown by the senior writer some years ago that a similar atypical epithelial growth of the uterine mucosa may be induced in the guinea-pig without the of estrogens. If one ovary is removed completely and the second partially, so that only a small ovarian fragment remains in the body ('' complete ovarian fragmentation "), a considerable change in the sexual cycle occurs: the follicular phase is prolonged and the vaginal mucosa may remain cornified for many weeks (Lipschutz, 5). Under these conditions a cystic glandular hyperplasia of the endometrium may occur in the guinea-pig.' When such animals are kept under observation for sev- eral years there is found in almost all of those in which the ovarian fragment has remained intact, a penetration of uterine glands bgtween the muscular coats of the myometrium. There may also be a cornification of the upper part of the cervix, where normally only mucified epithelium occurs. In one instance the aspect of the cervix was frankly precancerous (Lipschutz, 7).

1 This work has been aided by grants from the Ella Sachs Plotz Foundation for the Advance- ment of Scientific Investigation, and from Mr. Adolfo Eastman in Limache, Chile. 2 Similar observations were made also in the rat (Burch, Wolfe and Cunningham, 6) 185 186 A. LIPSCHUTZ, L. VARGAS, A. JEDLICKY, AND P. BELLOLIO

TABLE 1 : Efect of Eslradiol Injections on Uterine Weight - Estradiol hration o Body Weight (grn.) at and at End of Experiment Ester Injections and Uterine Weight (day81 Per Injt-c tion (pg.

Mono ben zoa te 85 0.1 3.7 385-660 600-820 795-950 . 1.2 1.o 3.4 Dipropionate 85 0.1 3.9 585-860 625-860 1.2 1.2 Benzoate- Bu t yra te 104 0.1 4.6 185-700 260-710 305-790 550-990 650-770 1.5 1.7 1.2 1.5 2.3 Monocaprylate 103 0.1 4.5 185650 260-790 305-800 560-830 770-890 1.4 1.9 2.1 1.2 4.2 __-___ -__ Benzoate 84 1 .o 37 440-585 4 7 5-680 490-580 575-670 595-670 2.5 2.3 2 .o 5 .O 2.3 Dipropionate 85 1 .o 3P 470-7 10 485-700 500-670 545-750 590-680 3.4 4.2 2.5 3.2 4.8 llenzoate-But yra te 97 1 .o 42 2 15-680 285-4310 3 10-660 570-980 620-920 3.0 2.5 2.0 4.5 3.7 Ca pryla te 96 1.o 41 235470 285-800 3 15-690 595-790 790-880 3.7 3.7 4.0 3.0 16.0 __

The ovarian fragment itself shows a pronounced tendency to form follicular cysts, sometimes with luteinization of the cyst wall. These results furnish convincing evidence that a prolonged follicular phase can cause atypical epithelial growth. The question remains, however, whether prolongation is of itself sufficient to produce this result or whether the quantity of estrogens produced in the unit of time must also increase, i.e. whether there must be in addition to prolongation of the folliculinemia a hyperfolliculinemia. Can atypical epithelial growth of the uterine mucosa be caused simply by a disturbance of the normal timing of the two endocrine phases of the ovary? This question is intimately bound up with another of considerable interest in experimental tumorigenesis. The carcinogenic substances, such as benz- pyrene, exert a tumor-producing action on or beneath the even when ap- plied in very small quantities. With the estrogens, on the contrary, large amounts seem to be necessary for a tumorigenic effect on the genital mucosa Can these two groups of substances, then, be aligned in respect to their tumor- producing properties? In the experiments to be described in this paper it is shown that a " tumori- genic " effect can be produced with only a few micrograms of estrogen provided certain conditions as to the timing of the application are observed.

EXPERIMENTS Thirty-five castrated female guinea-pigs were treated with different doses of 4 of estradiol-monobenzoate, dipropionate, 17-benzoate-3-n-buty- rate, and monocaprylate (8). Three subcutaneous injections were given weekly for three to three and a half months, Fifteen animals received 0.1 IAg. of esterified estradiol per injection, and 20 animals 1.0 pg. per injection, in 0.1 ml. olive . The total dose of estradiol in the first group of experiments ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 187 was between 3.7 and 4.6 pg. per animal in 85 to 104 days, whereas in the second group the total was 37 to 42 pg. per animal in 84 to 97 days. Treat- ment was begun seven to eight weeks after castration so as to be sure that the was in involution. The uterus of each animal was weighed and the genital tract was examined microscopically. A frontal section of the genital tract was made, showing the and cervix and the uterine horns (Fig. 1). Cross-sections of both horns at a distance of about 1.5 cm. from the cervix were also made. The observations are summarized in Tables I and 11. As seen from Table I, repeated doses of 0.1 pg. of estradiol in the form of the monobenzoate or dipropionate will maintain the uterine weight for several months not far from the normal level, which is about 1 gm. in the adult virgin guinea-pig. The " useful hysterotrophic dose '' of these esters, or the quantity

Transitional zone between cervix and uterus

Individual cervix

Common cervix Fornix ' Vaginal cervix Portio

Vagina

FIG. 1. SCIrEAlATlC DRAWINGOF FRONTAL SECTION OF BASEOF GUIXEA-PICUTERUS SHOWING VAGINA,CERVIX. AND BEGINNINGOF UTERINEHORNS of estradiol which when injected thrice weekly will maintain the uterine weight in the castrated guinea-pig on a normal level, is probably somewhat more than 0.1 pg.' In one of 5 animals the uterus weighed as much as 3.4 gm. Greater uterine weights were attained with 0.1 pg. of the benzoate-butyrate ester and especially with 0.1 pg. of the monocaprylate ester in experiments lasting about twenty days longer than those with the two esters first named. The maximum figures represent animals castrated after reaching adult life. Evidently, there- fore, the previous uterine history is of importance. With injections of 1 1J.g. the uterine weight is greatly increased, in general to about twice that observed in normal virgin animals. Exceptionally the in- crease is enormous, as in one animal of the monocaprylate group, with a uterus weighing 16.0 gm. The microscopic findings in the uterine mucosa corresponded with these observations on weight (Table 11). In the monobenzoate and dipropionate

3 In a new series of experiments with injections of 0.1 pg. a normal uterine weight was not always attained in the course of 82 days. 188 A. LIPSCHL~TZ, L. VARGAS, A. JEDLICKY, AND P. BELLOLIO

TAHLEI I : IIislologic Effrcts of Estradiol Injections (Figures intlicatc number of animals)

.-~ - Subniucosa With Metaplasis EY- of Uterus of Uterine Horn tra- Total (‘y6tic diol Nuni- Gland- - - With Metaplavia ~ __ Without Any per per ular between Cervix Metaplado In- of Hy- Abun- and IJterine With- of Uterine jec- Ani- dant Pig- llorn out With Horn tion mals pEa Vascu- Strati- lariza- nien Strati- fication (re.) tion fication __~- -______~_ Monobenzoate 0.1 0 0 0 Ol 5 Dipropionate 0.1 ;] ;* 0 0 0 01 Benzoate-B ut yrate 0.1 52 0 2 3 3 1 Monocaprylate 0.1 5 4ap.‘ 2 3 4 3 0 -- - ____-- Monobenzoate 1 .o 54 3 2 4 2 0 Dipropionate 1.o 5 Sap. 4 3 4 3 0 Benzoate-Butyrate 1 .o 5 Sap. 5 2 3 4 1 Monocaprylate 1.o 5 5 ap. 3 1 5 4 0

~ __ - * ap. = adenomatous polyps in two animals of the 0.1 pg. group and in most of the aninials of the 1 pg. group.

groups, with a single exception, the mucosa was normal following injections of 0.1 (Lg. Sometimes ( f in Table 11) small cystic glands were observed and there was dilatation of the blood vessels of the submucosa; but similar pic- tures are often seen in normal animals. In one animal only, with a uterine weight of 3.4 gm. (see Table I), there was a cystic glandular hyperplasia with the characteristic vascular changes in the submucosa. None of the 5 animals receiving 0.1 pg. of the monobenzoic and dipropionic ester per injection showed any sign of metaplasia of the uterine epithelium. The vagina re- mained open during the whole course of the experiment. Nevertheless, corni- fication of the vaginal mucosa was observed in only one instance; in general the vagina showed stratification but there was always a more or less pro- nounced leukocytic infiltration. In most of the animals receiving the same dosage of estradiol, i.e. 0.1 pg., in the form of monocaprylate for a longer period, there was, on the contrary, a thick cornification of the vaginal mucosa. The proliferation of epithelial layers at the entrance of the cervix also was considerable, or even enormous. Irregular solid cords penetrated deeply into the submucosa in 9 out of 10 animals of the monocaprylate and benzoate-butyrate groups (see Fig. 2B), producing a picture identical with that described by McEuen (9) as pre- cancerous in the rat. Similar solid epithelial cords were found also in the septum between the two cervices. Cornification sometimes reached the double cervix, where it is normally absent. In several of the animals of the monocaprylate group, and sometimes also in the benzoate-butyrate group, receiving injections of 0.1 pg., there was also a cystic glandular hyperplasia of the endometrium (Fig. 2A), though not al- ways of remarkable degree (Figs. 3A and 5A). The vascularization of the submucosa nearest to the endometrium was pronounced and pigment was sometimes present, a characteristic sign of former bleeding. Exceptionally the glandular hyperplasia may reach a degree so pronounced that adenomatous ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 189

FIG. 2. CHANCES FOLI.OWING SIIBCITTANEOZ'S INJECTIONS OF 0.1 &G. OF MONOCAPRYLATETHRICE WEEKLYFOR 103 DAYS(VII, 22) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 4.5 M. .4. Cross-section of uterine horn, showing highly developed cystic glandular hyperplasia. X 5. B. Frontal section of uterine base; portio near vagina; entrance into the vaginal cervix on the left. There is an enormous ingrowth of solid epithelial cords into the subjacent connective .

FIG. 3. CHANCES FOLLOWINGINJECTION OF 0.1 PG. OF MONOCAPRYLATETHRICE WEEKLY FOR 103 DAYS(VII, 18) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 4.5 PC. A. Cross-section of uterine horn, showing glandular cysts. X 5. B. Sudden transition from normal endometrium to metaplasia. X 110. C. Great extension of metaplasia; metaplasia of glands, also. polyps are formed (Fig. 2A); the connective tissue may disappear between the enlarged cysts which constitute these polyps. Another feature of importance in the monocaprylate and benzoate-butyrate group receiving 0.1 pg. of estradiol per injection was the metaplasia of the uterine mucosa. By " metaplasia " we understand two types of epithelial changes in the endometrium. This was sometimes of a pluristratified or pseudo-stratified type, as shown in Figs. 3B and C and especially in 5C and D; where almost solid cords are to be seen. Sometimes the cylindrical uterine epithelium was transformed into large clear cells, probably mucified (Fig. 4B), FIG.4. CHANGESFOLLOWING INJECTION OF 0.1 pc. OF MONOCAPRYLATETHRICE WEEKLY FOR 103 DAYS(VII, 21) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 4.5 w. A. Cross-section of uterine horn, of almost normal aspect. Uterine weight 1.2 gm. X 5. B. To the left, normal glandular epithelium; to the right, gland with large clear cells, prob- ably mucified.

FIG. 5. CHANCES FOLLOWINGINJECTION OF 0.1 CCC. OF 17-BENZOATE-3-zj-BUTYRATE (VII, 2) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 4.6 CIC. A. Cross-section of uterine horn, showing glandular cystic hyperplasia. X 5. B. Sudden transition from normal endometrium to metaplasia. C. Pseudo-stratification of endometrium. X 220. D. Uterine glands almost completely solidified.

190 ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 191

FIG. 6. CIIASGES FOLLOWISGISJECTIOS OF 1.0 PG. OF DIPROPIONATETHRICE WEEKLY FOR 85 DAYS (VI, 23) ; TOTALQUANTITY OF ESTRADIOI.ADMINISTERED 38 PG. Uterine weight 3.4 gm. Cross-section of uterine horn. Adenomatous polyp occupying great part of uterine cavity. X 5.

FIG.7. CHANGESFOLLOWING INJECTION OF 1.0 ICC. OF MONOCAPRYLATETHRICE WEEKLY FOR 96 DAYS (VII, 27) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 41 PC. A. Cross-section of uterine horn. Enormous development of cystic glandular hyperplasia. x 5. B. Frontal section of uterine base. Transitional zone between upper part of individual cervix and uterine horn. * Solid epithelial cords to the right, and nearby glandular cysts of uterine horn, very similar to those present normally in the cervix and previously found in the uterus in experiments with ovarian fragmentation (Lipschutz, 5, 7). These changes occurred only in limited areas. In general the change from normal cylindrical epithelium to metaplasia was abrupt (see especially Figs. 3B and SB). It is remarkable that metaplasia was present in an animal of this group which attained a uterine weight of only 1.2 gm. (Fig. 4), the normal cells of the endometrium being replaced by large clear cells (Fig. 4B). These results show beyond question that with a total quantity of 4.5 pg. of estradiol in the form of the benzoate-butyrate or, more especially of the monocaprylate, given in 45 injections over a period of three and a half months, atypical epithelial growth is often established. 192 A. LIPSCHUTZ, L. VARCAS, A. JEDLICKY, AND P. BELLOLIO

FIG. 8. CHANCES FOLLOWINGINJECTIOH OF 1 M. OF MONOCAPRYLATETHRICE WEEKLY FOR 96 DAYS (VII, 23) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 41 ~rc. A. Cross-section of uterine horn. Polyp in the uterine cavity. Cystically enlarged glands. x 5. B. Stratified epithelium of endometrium and glands. X 45.

With 1.0 IJg. per injection all these phenomena become accentuated (Ta- ble 11). Almost no animal receiving this larger dose escaped formation of adenomatous polyps (Figs. 6, 7A, 8A) and metaplasia (Figs. 8B, 9). The adenomatous polyps of the uterine horn often extended down into. the cervix (Fig. 10). Such polyps may originate from the transitional zone between the individual cervix and the uterine horn, or from higher parts of the horn (Figs. 10 and 11). Sometimes uterine and cervical polyps are found nearby in the cervix. With greater quantities of esterified estradiol, as 5 or 10 pg. per injection, the adenomatous uterine polyps may assume voluminous proportions (Figs. 12, 13, 14), and may descend even into the vagina (Fig. 15). The transitional zone between cervical and uterine mucosa is of especial interest as to metaplasia. Under normal conditions the two types are sharply delimited, with an abrupt transition from the mucified epithelium of the cervix to the cuboid or cylindrical endometrium, though glands with mucified and cylindrical epithelium are to be found near by. In our experiments with injections of 0.1 pg. and especially 1.0 pg. of the benzoate-butyrate and mono- caprylate the transitional zone became the seat of a very pronounced meta- plasia. The cylindrical epithelium of the endometrium and of its glands may be replaced in certain areas by large clear cells or by a stratified epi- thelium. The glands in this zone were often transformed into solid epithelial cords (Fig. 7B). DISCUSSION The experiments recorded above give full evidence that atypical growth of epithelium can be established with infinitesimal quantities of estrogens. With a total dose of 4.5 pg. of esterified estradiol (monocaprylate, benzoate- butyrate), and especially with total doses of 37 to 42 pg. of the different esters, injected over a period of three and a half months not only was a cystic glandular hyperplasia produced but there appeared also adenomatous polyps and a pluristratified metaplasia of the endometrium with transforma- tion of uterine glands into solid cords. Likewise, solid epithelial cords pene- ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 193

FIG.9. CHANCES FOLLOWINGINJECTIOX OF 1.0 pc. OF MONOBENZOATETHRICE WEEKLY FOR 84 DAYS (VI, 3) ; TOTALQUANTITY OF ESTRADIOLADMINISTERED 37 ~rc. Uterine weight 2.3 gm. Cross-section of uterine horn. Pluristratified metaplasia of endo- metrium. Sudden transition from endometrium of normal aspect to a pseudo-stratified island. Uterine glands of normal aspect in the submucosa.

FIGS.10 AND 11. ADENOMATOUSPOLYPS DEVELOPING AFTER INJECTIONS OF MONOBENZOATE ASD DIPHOPIONATE Fig. 10. Animal treated with 1 F". of monobenzoate thrice weekly for 84 days (VI, 5). To- tal quantity of estradiol administered 38 pg. Frontal section of uterine base. Adenomatous polyps descending into both individual cervices. F = fornix. Fig. 11. Animal treated with 1 PK. of dipropionate thrice weekly for 85 days (VI, 21). To- tal quantity of estradiol administered .%?fig. Frontal section of uterine base. Adenomatous polyps with large glandular cysts, especially on the right, descending into the individual cervix. trated into the wall at the entrance of the cervix. We are as yet unable to say whether the atypical growth of the uterine epithelium produced by such small quantities of estrogen is irreversible as is that induced by greakr quanti- ties (Allen et al, 4; Lipschiitz, Iglesias and Vargas, 3). How is the '' tumor-producing " action of small quantities of estradiol to be explained? It may be supposed that atypical growth and tumorigenesis are 194 A. LIPSCBUTZ, L. VARGAS,A. JEDLICKY, AND P. BELLOLIO due to the maintenance of a constant circulation of estrogens in the organism. As shown by Miescher and coworkers (8), the benzoate-butyrate ester and the monocaprylate ester of estradiol prove in the Allen $nd Paisy test to be even less active than the monobenzoate and diprogionate. ky On the other hand, the first-named esters have a prolonged action in the body. {Evidently when such esters are injected thrice weekly, a stable. folliculinemia {is more easily estab- lished, and this we consider as the principal factor in ithe tumor-producing action of the estrogens. I It is well known that follicular hormones when introduced into the body by subcutaneous or intravenous injection disappear rapidly from the blood by

FIGS. 12 AND 13. CHANCES FOLLOWIKG.4DhII~ISTRATION OF 5 AND 10 PC. OF DIPROPIONATE Fig. 12. Animal treated with 5 ~g.dipropionate thrice weekly for 90 days (VI, 27). Total quantity of estradiol administered 195 pg. Frontal section of uterine base. On the left, uterine polyps with glandular cysts; parts very rich in connective tissue. Other polyps descending into the individual cervix. On the right, uterine polyps descending into the common cervix close to the vaginal cervix. Distal part very rich in blood vessels. Fig. 13. Animal treated with 10 pg. of dipropionate thrice weekly for 87 days (VI, 31). To- tal quantity of estradiol administered 380 pg. Enormous uterine adenomatous polyps, especially on the right. The distal part of the polyp is inverted close to the upper part of the common cervix, evidently having encountered an obstacle to its descent. inactivation, estradiol and being transformed into , which com- bines with (Marrian et al, 10). In the Allen and Doisy test the activity of the glucuronic ester of estriol is probably only about one two- hundredth as great as the activity of free estradiol, even with maximum figures for estriol (Emmens, 11). The transformation of estradiol or estrone into estriol and the combination of the latter with glucuronic acid can reasonably be interpreted as a fundamental means of self defense against epithelial tumori- genesis. On the contrary, esterification with benzoic, propionic and other acids in some way protects the injected estradiol against rapid inactivation. It is remarkable that a different hormone threshold is necessary to produce epithelial and fibrous growth respectively. Uterine fibroids (Nelson, Mori- card and Cauchoix) and similar extra-uterine tumors (Lipschutz, Iglesias, ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 195

Vargas and others), as is well known, can be induced in guinea-pigs by pro- longed injections of estrogens. These uterine and extra-uterine fibroids may appear when 5 to 10 pg. of estradiol per injection are given in esterified form thrice weekly for three months. The tumorigenic threshold for fibroids dif- fers with different esters; so far the monocaprylate ester has proved to be more tumorigenic than the three others which we have examined (Bellolio, 12; Jedlicky, Lipschiitz and Vargas, 13). With 1 pg. of esterified estradiol per injection uterine fibroids visible to the naked eye were found in only one of 5 animals receiving the monocaprylate ester and in none of 15 animals receiv- ing the other esters. On the contrary, as recorded above, injections of 1 pg.

FIGS. 14 AND 15. CHANGESFOLI.OWING ADMINISTRATION OF 10 w. OF DIPROPIONATE Fig. 14. Animal treated with 10 ~g.of dipropionate thrice weekiy for 87 days (VI, 34). To- tal quantity of estradiol administered 380 ~g.Adenomatous uterine polyps on both sides. On the left side polyp approaching or entering the common cervix. Fig. 15. Animal treated with 10 pg. of dipropionate thrice weekly for 87 days (VI, 32). To- tal quantity of estradiol administered 380 ~g.Enormous adenomatous uterine polyps descending into the vagina. induced epithelial metaplasia in the uterus in 19 out of 20 animals (Table 11), and some kind of epithelial metaplasia was produced with 0.1 pg, in 9 out of 15 animals. Fibroids were never induced with this smaller amount. The present findings offer an opportunity to discuss the problem of the graded reactivity of the different parts of the genital tract to follicular hor- mones. Loeb (14) was the first investigator to recognize the importance of these gradations for a proper understanding of the varied microscopic estrous changes which take place in the mucosa passing from the vagina to the uterus. The senior writer (15) has tentatively admitted that the classical conception of seuils difldrentiets of PCzard applied to the different reactions of the vaginal, cervical, and uterine mucosa to estradiol may be of use in explaining the dif- ference in frequency of pluristratified cancer at the entrance of the cervix and in the uterus in women. Under normal conditions, as is the case in estrus in the guinea-pig, the vaginal mucosa and the adjacent parts of the 196 A. LIPSCHUTZ, L. VARGAS, A. JEDLICKY, AND P. BELLOLIO

cervical mucosa reveal pluristratification and cornification, or ('epidermiza- tion," whereas the upper part of the common cervix and the entire individual cervix present mucification, and the endometrium a unique layer of cylindrical epithelium. Experimental observation shows, however, that the upper parts of the genital tract also are capable of epidermization; all depends on the quantity of the hormones and on the time during which they act on the mucosa. When the follicular phase is artificially prolonged by ovarian fragmentation in the guinea-pig, pluristratification and cornification extend into the upper parts of the common cervix and even into the individual cervix, where normally mucification occurs (Lipschutz, 7). Our present work with prolonged injec-

FIG. 16. CHANCES FOLLOWIKCINJECTION OF 40 W. OF BENZOATETHRICE WEEKLY FOR FOUR MONTHS (I, 19) ; TOTALQUANTITY OF ESTRMIOLADMINISTERED 2120 CC. Pluristratified metaplasia of endometrium of a type similar to that in the vagina but without cornification. In some parts there is mucification of superficial layers which so often occurs in the vaginal mucosa of the guinea-pig. * Normal endometrium. X 90.

tion of estrogens shows that under the same quantitative and time conditions by which cornification is established in the upper part of the common cervix and at the entrance of the individual cervix, there is in the endometrium only metaplasia with mucification; pluristratification, when it occurs, is seen in limited areas only. This pluristratified metaplasia offers in general a quite different aspect-" pseudo-stratification " (Figs. 3, 5, 8, 9)-than the pluri- stratified epithelium in the vagina and in the cervix, But eventually, though rather rarely, when injection of follicular hormones is continued for many months the endometrium may be partially replaced by a pluristratified epi- thelium which is very similar to though not identical with that in the vagina (Fig. 16) ; in the guinea-pig cornification was never seen by us in the uterine horns proper. All this is in favor of our suggestion that the graded reactivity of the genital tract to estrogens may help in an understanding of the predominance of the pluristratified type of cancer at the entrance of the cervix and that of the cylindrical type in the uterus itself. There is indeed in the guinea-pig, as ATYPICAL EPITHELIAL GROWTH OF UTERINE MUCOSA IN GUINEA-PIG 197 shown in our work, one exception to the rule: the transitional zone, or the proper limit between the cervix and the uterine horn, is more ready to react with pluristratification than the individual cervix below. But it must be borne in mind that there are probably very considerable species differences in the capacity of tissues to react to follicular hormones.

SUMMARY Atypical epithelial growth of the endometrium can be induced in the castrated guinea-pig by as little as 4.5 pg. of estradiol when the latter is administered in the form of certain esters (monocaprylate and 17-benzoate-3- n-butyrate) in doses of 0.1 pg. over a period of three and a half months. Pluristratified metaplasia in limited areas of the endometrium is sometimes induced by these small quantities of esterified estradiol. These changes became more apparent with a total of 37 to 42 pg. of the different esters given in doses of 1 pg. over a period of three months. Under these experimental conditions adenomatous polyps also are formed at the expense of the proliferated uterine glands. The polyps may occupy the whole cavity of the enlarged uterus. With sufficient quantities (1 to 10 pg. per injection, or a total quantity of 37 to 370 pg. during three months) these polyps may descend into the cervix and even into the vagina. The tumorigenic threshold of esterified estradiol is lower for atypical epi- thelial growth than for fibroids. It is suggested that the tumor-producing action of estradiol on the uterine epithelium may depend on a stable folliculinemia, which evidently is easily maintained by injecting estradiol in an esterified form. The rapid transformation of free estradiol and estrone into estriol in the organism of the woman and the combination of estriol with glucuronic acid can be considered as an important means of self defense against the tumor- producing action of these physiological substances. The graded reactivity of the genital mucosa to estrogens, which diminishes in the direction of the uterus from the vagina, may explain the difference in frequency of the pluristratified type of cancer at the entrance of the cervix and the cylindrical type of cancer in the uterus proper in women. NOTE:Our thanks are due to Doctor Karl Miescher of Messrs. Ciba in Basel, Switzer- land, for a liberal supply of different esters of estradiol used in this work.

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