Clinical experiments with diethylstilbestrol

PALMER

:

EXPERIMENTS

\VITH

DIE:TH~I,STILB~STROI,

Sijl

5. Puerperal infection was no more frequent in the group with positive vaginal culture than in women with negative vaginal culture. 6. Isolation of the gas bacillus from the vagina in three cases, either during labor or in the puerperium, would demonstrate that the recovery of this organism from the vagina does not necessarily imply infection with the Welch bacillus, since all three patients remained afebrile and presented no evidence of puerperal infection. 7. The above results would indicate that Cl. udchii may be fount1 normally in the vagina of the adult pregnant fernall:, but that it is noI necessarily a permanent inhabitant. 8. Recovery of the gas bacillus from the normal vagina is not, neccssarily of serious import, but in the presence of traumatized or d&talizctl t.issue,srrious infection wit,h the gas bacillus may result. REF’ERESCI’S

(I) kfanahan,

, 1.

l’ohlman, IT. R.: d. ;\. M. A. 109: 254, 1937. (2) Sa&&, J. F., and C. P.: J A M. A. 113: 14, 1939 (3) Cosgroue, N. -I., and Ilarry, T. il.: England J. Med. 222: 344, 1940. (4) Stander, H. 6.: Williams’ Obstetrics.

New A Textbook for the Use of Students and Practitioners, ed. 7, New York, 1936, I). Appleton-Century Co., pp. 1170-1171. (5) Cz~rtis, d. H.: Obstetrics and Gynecology, ed. 1, Philadelphia, 1933, W. B. Saunders and Co., Vol. II, p. 204. (6) Wrigley, A. J.: Proc. Roy. Sot. Med. 23: 1645, 1930. (7) Schottnziiller, H. : Miinchen med. Wchnschr. 57: 1817, 1910. (8) Falls, F. H.: AM. J. OBSF. & GYNEC. 25: 280, 1933. (9) Bysshe, S. M.: Ibid. 35: 995, 1938. (10) Society of American Bacteriologists, Manual of Methods for Pure Culture Study of Bacteria (Committee on Bacteriological Technic), 1936, and The Study of Obligately Anaerobic Bacteria, ed. 3; Geneva, N. Y., Society of American Bacteriologists.

(11,INJCAL EXPERIMENTS

AIUN (From

the Gynecological

PALMER,

WITH

M.D.,

Endocrine

SAN

Laborntory

I)IETI-E~‘,STII,BESTROJ,”

FRANCISCO, and

(:liSc

Cxr,r~.

lkp~futment

of

Obstrtr&~s

L’CKERMAN” has introduced the theory of a ut’zrine threshold to estrogen into the picture of the menstrual mechanism of the rhesus monkey. He writes: “The concept of an oestrone threshold of the uterus in primates is open to at least three interpre!;ations. It mig!lt mean the least amount of hormone necessary to produce, in the shortest, possible time, definite signs of endometrial growth in a spayed monkey. It might mean the least amount of hormone, given over a stated number of days, that is needed to influence the endometrium sufficiently for thth cessation of injections to be succeededby uterine bleeding. Or it might. mean the smallest dose which, given daily for an indefinite period, will maintain the endometrium without uterine bleeding occurring. ’ ’ Zuckerman’s experiments are concerned primarily with the second interpretation. In effect, bleeding occurs when the uterine threshold rises above the amount of estrin present, or when the amount of estrin present falls

Z

*This

study

was

made

possible

by

the

Christine

Breon

Fund

for

Medical

Research.

S62

AMERICAN

JOLJRK.\I.

OP

OBSTE:TRICS

.\Kl,

~:YiXE:(‘OI,OGY

below the uterine threshold. In other words, t,hc: relative levels of these two factors with respect to each other seem IO be the state which, if it could be measured, determines the presence or absence of uterine bleeding. Zuckerman”* 6 has produced uterine Heeding in the ovariectomized monkey in two ways. By varyin, v the manner in which estrone is administered, cyclical phases of uterine bleeding were induced, reflecting on the one hand the fluctuation of the estrin level and on the ot,her the fluctuation of the uterine threshold. In order to simulxtc Flwtuation of Estrin Level in the Monkey.-(Zuckerman.(~j what is generally believed to be the normal production of e&in by the ovary, it. was necessary to supply estrogen in increasing and suddenly decreasing amounts of the hormone; when the latter is given in increasing amounts in a fonrlrr~~-day period,

250

Fig.

l.-Threshold

cycle

in monkey

produced

with

e&one.

(Zuckerman,

1937.)

beginning with the onset of menstruation, the treatment is comparable to the time fol Estrin produces its positive morphologic growth and rupture of the graafian follicle. effects in the endometrium during the first half of the normal cycle in the rhesus monkey as well as in the human female. To simulate the sudden fall of estrin level that occurs following the rupture of the Graafian follicle, the daily inject,ions of the hormone were stopped after the fourteenth day. An optimum amount (experimentally) of estrone required to influence the endometrium of a monkey in fourteen days so that cessation of injections would be followed by uterine bleeding was found to be 2.7 mg., administered as shown in Fig. 1. Bleeding occurred regularly on the twenty-second or twenty-third day of the “artificial” cycle or tight to nine days after cessation of injections, indicating that, the cessation of therapy on the fourteenth day was followed by a fall in the level of estrin below the uterine threshold on about, the twenty-second or twenty-third day. The uterine bleeding thus produced was a reflection of a fluctuating (falling) estrin level. In order to lengthen the artificial cycle from twenty-two to twenty-eight days, a daily ~ubthreshold dose of estrogen was started on the fifteenth day which was sufficiently small to allow the estrin to fall more gradually to a level below the uterine threshold and sufficiently great to prevent, the uterine threshold, if fluctuating, from The daily dose of e&one rising above the estrin level before the twenty-eighth day. necessary for this purpose in the monkey was foucd by Zuckerrnan to be 10 to 13 gamma (Fig. 2). As soon as bleeding began the animal was started on a new cycle. The production of an artific.ial cycle constitutes the nearest approach to the way in which the ovary produces its effect during an anovular cycle that has yet been recorded. The

PALMER

:

ESPERIMEFTS

WITH

I~I~TIIYI~S’I‘II,RI:S’l’R~~i~

iiG1

ovulatory rycle in which progestin takes part has also heen simulated and pro~lur~l artificially (Zuckermani) ; this particular issue is not. however, relevant to ile rlinical experiment under discussion. Flzcctuation of Uterine Threshold in the Xo?zkry.-The fluctuating uterine thr+4lold has been demonstrated by Zuckermanj in an ovarieetomized monkey whil,lt was injected daily for :?fiS days with the same amount of estrone (lot gamma per tl:t,v 1. In spite of the repeated injections, eight separate phase. q of bleeding occurred rliiring the experiment but at intervals varying from tivc to seven weeks. In view of’ tile fault that uterine bleeding will not OCCUR in spayed monkey:? under continuous ~II,jection with an amount of hormone that is well above threshold value (100 gamnm per dayj, the successive phases of bleeding ob.rerred in this monkey must be interpreted as reflections of successive falls in the degree of cstrogenie *t,imulation experienced by the animal or successive rises of the uterine thre&old. Since tht monkey was being continuously injected with the same amount, of hormone daily. it follows that rhythmical changes of a kind that decreased the effectivity of tht> hormone or.eurred within the animal itself. The possibility that the adrenals and tile pituitary are in some way concerned is being investigated. 300

0 t

2

4

6

8

16 18 10 12 14 DAYS OF TREATMENT

20

a?

24

ARTIFICIAL

produced subthreshold

CYCLES

IS THE

Length with estrone. dose of estrone from

HLTMAN

28

30

BLEEDING

BLEEDING

Fig. %-Threshold cycle in monkey longed from 22 to 28 days with daily forward. (Zuckerman, 1937.)

26

of cycle flfteenth

lx%day

REISG

I have used diethylstilbestrol in the human being to induce cyclical estrogen withdrawal bleeding. Wenner and JoEI and Wenner3 report that a minimum total dose of 25 mg. of diethylstilbestrol orally is rcquired to produce what appears to be normal endometrial growth, while a dose of 60 mg. or more may produce endometrial hyperplasia. Thc~ availability of 1 and 5 mg. tablet,s of diethylstilbestrol resulted in the a,doption of the following p Ian for the production of an artificial estrogenic cycle in the human subject : 1 mg. per day for seven days followed by 5 mg. fier day for seven days. Thus one (7oursc of therap? would amount to a total dose of 42 mg. The treatment of 4 patients with primary amenorrhea in ally followed by uterine bleeding after a latent interval of five Prolongation of the latent interval was effected in 2 artificial subthreshold dose of 0.1 or 0.3 mg. of diethylstilbestrol daily onward. The pH of the vaginal mucotia before and during is shown in the last two columns of Table I. Figs. 3 and structurr of the endometrium obtained by biopsv from Patient

tl;is manner was IISIIor six days (Table I). cycles by giving :L from the fifteent.11 (la? each cycle of therapy 4 show the histologic. 11. 1’. on the first ant1

864

AMERICAS

JOUR;\ThI,

OF

OBS’CETRI~T

hSt)

GYSI?A’OJ>O(;Y

fourteenth days, respectively, of an artificial cycle. Fig. 5 shows a mild degree of cystic glandular hyperplasia produced after the third attempt to produce withdrawal bleeding in Patient M. M. The total dose of diethylstilbestrol used for this cycle was 70 mg. These histologic findings are in agreement with the report of Wenner and Joel,4 that more than 60 mg. of diethylstilbestrol may produce hyperplasiw. Forty-two milligrams have been an optimum dose of diethylstilbestrol for the production of normal endometrial proliferat,ion in fourteen days. Estrogenic hormone x-as absent from the urine of the 4 patients listed in Table I when treatment, was

Fig.

3.-Photomicrograph artitlcially

of inactive endomctrium produced threshold cycle

Fig. 4.-Photomlcrograph fourteenth day of an artIflclally of 42 mg. diethylstilbestrol eleven days later (X120).

of was

normal produced given.

proliferative threshold Bleeding

in

obtained Patient

at M. P.

endometrium cycle in Patient occurred from

the beginning (X130.1

of

an

obtained on the M. P. A total dose this endometrium

PALMER

:

EXPERIMESTS

WITH

DIETH~I,~rII,BESTROI,

SG5

started. To date there has been no spontaneous occurrence of menstruation follotring substitution therapy, other than the immediate withdrawal bleeding. Variations in the administration of diethylstilbestrol were tried in 4 postmenopausal patients whose pelvic organs were intact, (Table II!. It is noted ( 1) that the lengths of the artificial estrogenic cycles varied with l,he duration of therapTvhen t!re daily dose was 1 or more mg.; (2) that when the daily dose was decreased 10 0.1 to 0.3 mg. bleeding occurred during therapy although the latent interval IXtween the fall of daily dose and the onset of bleeding was p,rolonged; (3) that the latent interval between complete cessation of therapy (when the daily dose was I mg. or more) and the onset of bleeding was consistently five to six days; and (-I- J that the pH of the vagina was lowered in every instance following estrogen therapy.

Fig.

5.-Photomicrograph total oral dose

of

of cystic endometrium 70 mg. diethylstilbestrol

obtained from in fourteen

Patient days.

M. M.. after r X120.)

:L

DISCUSSION

The purposes for which the clinical experiments relborted in this paper were intended are : (1) To ascertain the oral t,herapelltic dose of diethylstilbestrol necessary for the production of normal cndometrial prdliferaovarian tion and estrogen-withdrawal bleeding in women without function. (2) To estimate the amount of diethylstilbestrol administered by mouth over a period of fourteen days which would produce abnormally hyperplastic endometrium. (3) To produce, by cyclical estrogen therapy, artificial menstrual cycles in women according to Zuckerman ‘s interpretation of “threshold cycle ” as it pertains to the monkey. (4) To demonstrate the presence or absence in the human being of a fluctuating uterine threshold to diethylstilbestrol. It, is admit,ted, for the present, at lea.&, that probably there is no particular reason for inducing ut,erine bleeding by substitutional therapy. However, definite growth of the uterus and development of

- ZZZ DIAGNOSIS OF AMENORRHEA

PT.

II.

P.

ICE

c

&GLE DAYS

--

Primary (genital hypeplasia)

42 7 42 42 0.::

mg. in 14 mg. in i mg. in 14 mg. in 14 mg./day 22nd dav 42 mg. in”.14 11.1

G. A.

Primarv (pituitary infantilism) __D. S:. Primary (genital hypoplasia) M. M.

,ENGTII Clm

‘PREATME?rTT ( DIETHYLSTILBESTROL)

12 42 42 19 7

mg./clay

2nd day mg. in I4 mg. in 14 rng. in 14 ma. in 19 mr. in 7

days days day.% days 15tlt-

20

days lv%lr-

21

days days tl:tys tlays clays

I!) 19 24 21

20 25

-

Primary (genital lrypoplasia)

*Data ethylstilbestrol.

pertainin:;

19 to

1.

u.

Bleeding %ul day (luring treatment No hlreding 5 days 5 days 5 days No hleedine No bleedinff Bleeding 22ml day during treatment No bleeding No hleeding .? days

4.0 -1.4 ti.0 4.x 4.4

TATENT INTERVAL BFT1 . ~‘ESSA’I’ION 0~’ TtiEATMENT AND BLEEDIN\‘(;

Cl F

__~

-.

L

.431ENORRllE.4

r)H

BE] DUR-IKG FORE PREATTREAT-

ci days No bleeding fi days :t days

thl

DIAGNOSIS

PT.

VAGINAL

LATENT INTERVAL BET. CESSATION OF TREATMENT AND BLEEDING

I

4.0 4.0 4.0 -

7.1; G.8

5.2

Ii.0

4.0 -

-r 6.0 j.6 5.6

4.0 4.0 -

wing

-

wit11

tli-

-i‘I

Secondary (natural

J. II.

J.

N.

menopause 3 years) Secondary ( IldtUKil menopause 30 years) Secondary (natural

.5 days Ii days .? dityS Ii days No hleeding No bleeding Ii clays 6 days Bleeding 3lst day during treatment 3 days

IIleIlOpZtUW

ti years) r1.1 nYg./axy 31st day V. N.

I utit-

Secondary (natural menopause 5 years)

.‘Lam pertammg to

ethylstilbestrol.

th resho

I cycles

produced

artificial

in

the

human

6.8 4,s

-

44 4:r, 4.0 4.0 4.8

52 .: 4.0 5.0 4.8

--

4.4 -

being

with

di-

other secondary sexual characteristics were effected by cyclical estrogen therapy along with periodic uterine bleeding. There is considerable experimental evidence that prolonged uninterrupted estrogen treatment produces undesirable results that are not produced by cyclical therap) (Palmer*). The importance of cyclical est,rogen therapy has been revealed by an investigation of the ease history of Patient M.P. (T,able I). For seven and one-half months this patient received thrice-weekly injections and oral doses of one estrogen or another wit,hout periods of observation for withdrawal bleeding. Efforts had been direct.ed .toward the development of mammary tissue for cosmetic reasons and t,he establishment 01’ i;he menarche. Withdrawal bleeding of two days’ duration occurred finally six days after the cessation of the long uninterrupt,ed course oi estrogen therapy. Following the first, attempt at cyclical t,hcrapy, withdrawal bleeding occurred six days after a threshold cy-clr of tlicthylstilbcst,rol, and endured four days.

1. Zuckcrman’s experimental data on ~,hc pt’oduct~ion of artificial menstrual cycles and a fluctuating uterinc t~hrcshokl lo cstrone in 111~~ monkey are reviewed. 2. Artificial menstrual cycles (diethylst,ilbestrol withdrawal bleeding) were induced by cyclical diethylstilbestrol therapy in 4 patients with primary amenorrhea and in 4 postmenopausal women. 3. Forty-two milligrams were found to be an op;timum oral dose of diethylstilbestrol which in fourteen days would produce a normal degree of endometrial proliferation and would be followed by uterine bleeding. 4. Seventy milligrams of diethylstilbest~rol by mouth in fourteen days produced a slight degree of cystic glandular hpperplasia of the entlonietrium. 5. The consistency of the latent interval that precedes withdrawal bleeding in the group of primary and postmenopausal amenorrheic patients, regardless of variation in dosage and manner of administrai.ion. leads one to believe that a fluctuating uterine threshold to estrogen may not be a factor with which to be concerned as it will he shown 1.0 be in patients with uterine bleeding (Palmer? 1, and as if is known to be in the ovariectomized monkey. The diethylstilbestrol used in this investigxtion E. R. Squibb Companies.

vxs supplird by the ITli Lilly :1ni1

REFERESCES (In press.) (2) Id em: Anr. .J. ORF~. & (:YSE(‘. 41: June, 1941. Schweiz. med. W’rhnschr. 70: 277, 1941). (4) ?Tenner, II., and ,7&l, Ii.: Lancet 2: 688, 1939. (5) Zuokerm.nn, 8.: Pror. Ilc!y. Sot., Ser. B., 123: (7) Irlm,: Jhd. R., 124: 150, 19.:7. 441, 1937. (6) I&m: Ibid. B., 123: 457, 193’7. (1)

(3)

(A

Palmer,

Wennrr,

swont7

A.:

II.:

contributions

to

this

su,bjwt

will

nppenr

in thr

.Tun.r

~SSUP

of

thr-

JOITRNALJ