Estrogen metabolism in normal and neoplastic endometrium E. GURPIDE L. TSENG* S. B. GUSBERG Nrw

York,

Nrul York

Studies on normal endometrium at different phases of the menstrual cycle have shown that progesterone and synthetic progestins reduce the levels of estradiol receptors in the tissue and increase the activity of estradiol-17p-dehydrogenase, an enzyme that converts estradiol to estrone. These effects may account for the antiestrogenic characteristics of the progestins. Similar effects were obtained in some postmenopausal patients with endometrial adenocarcinoma treated for two to 10 days with oral medroxyprogesterone acetate. These results point to the potential usefulness of a short-term, in vivo biochemical test which, combined with histologic observations, may identify patients who are likely to respond to treatment with progestins. (AM. J. OBSTET. GYNECOL. 129: 809, 1977.)

THE ISOLATION OF naturaI estrogens by Allen and Doisy, in 1923, led shortly to their therapeutic use, and the synthesis of diethylstilbestrol by Cook, Dodds, and Hewitt, in 1937, made available an inexpensive oral preparation for application to a myriad of symptoms. Soon it became apparent that long-term administration of estrogen without progestational modification could produce atypical hyperplastic proliferation of the endometrium in human subjects, in a manner identical to that seen in women with failure of ovulation;’ further, it was suggested that these endogenous and exogenous factors placed these women at higher risk for later development of endometrial cancer. This circumstantial and morphologic evidence of hormone sensitivity2 awaited the advance of steroid metabolic technology in the 1960’s for rationalization on a biochemical basis. Increased conversion of the adrenal precursor andro-

stenedione to estrone in obese postmenopausal women3a4 and in women with endometrial cancer’ was established in the recent decade. Careful epidemiologic studies+* most recently confirmed an increase in risk for endometrial carcinoma in postmenopausal subjects during long-term estrogen administration. Progress in this area has been summarized elsewhere.” These considerations together with the efficacy of progestin in the treatment of some patients with advanced endometrial cancer”’ I’ suggested the importance of studying the tissue metabolism of endometrial neoplasms after the administration of a progestin. Prior in vitro investigations provided information on levels of estradiol receptor (E,R)‘* and estradiol-17/3dehydrogenase (&DH) activity,i3 against which similar measurements in abnormal endometrium could be compared. These parameters are relevant because they affect the intracellular concentration of estradiol (E2) and its biological action. Furthermore, these studies on normal endometrium revealed an influence of progesterone on E2R and EtDH levels which suggested a possible key to the understanding of its therapeutic use in adenomatous hyperplasia and endometrial carcinoma.

From the Departments of Obstetrics and Gynecology and (If Biochemistry, Mount Sinai School of Medicine, City l’niuersit?, of Nest York. Supported by Grants CA-15648 National Institutes of Health.

and HD-07197

from the

Presented at the One Hundredth Annual Meeting of the .4merican Gvnecoloeical Societv.i. Litchfield Park. Arizona. i .4pri113-16, 1977:

Methodology

Reprint requests: Dr. S. B. Gwberg, The Mount Sinai Hospital, I1 76 Fgth Ave., New York, New York 10029. *Present address: Department of Obstetrics and Gywcology, Health Sciences Center, State University Nm York, Stony Brook, New York.

Intracellular EzR concentrations were estimated by incubating tissue slices with an excess of tritiated estradiol and measuring the amount of specifically

of

809

810

Table

Gurpide, Tseng, and Gusberg

I. E?R levels

2 Normal Proliferative After progestin administration Secretory Early (days 17- 19) Middle (days 20-23) Late (days 24-28) Hyperplastic (postmenopausal) Adenocarcinoma (postmenopausal) Well-differentiated Exogenous estrogen users Poorly differentiated

39 9

3.1 1.5

1.3 0.7

; 2

21.: 0:5

0.5 0.3 -

7

2.8

0.6

17 4 3

2.3 3.8 2.0

1.1 0.8 1.5

(From Gurpide and associates.2’ Published with the permission of theJolcr?lal of Steroid Biochemistq.) bound hormone in nuclei isolated after homogenization of the tissue.” EZR levels were expressed as picomoles of ES per milligram of deoxyribonucleic acid (DNA). E2DH activity was determined by measuring the rate of formation of labeled estrone (Et) from tritiated Et added to tissue homogenates, under conditions of excess substrate and cofactor.‘3 Enzymatic activities were expressed as nanomoles of Et formed per milligram of protein x hour.

Results EzR. Table I illustrates the results obtained by measurement of l$ receptor levels in various endometrial tissues. It is noteworthy that the levels of receptors are high in proliferative endometrium and decline during the secretory phase, as previously reported,‘* a finding consistent with results from other laboratories.“‘-“* The decline in the receptor level in the luteal phase is due to the influence of progesterone, as the administration of progestins to patients in the follicular phase of the cycle resulted in lowering of the E2R levels seen in proliferative endometrium from untreated subjeckZ3 Similar effects of progestins on l&R levels have been observed in genital tract tissues of other mammalS.?“-‘6 Hyperplastic endometrium obtained from perimenopausal and postmenopausal patients had E2R levels comparable to those of proliferative endometriurn. The EtR levels in specimens of endometrial adcnocarcinoma in postmenopausal patients were lower than those in proliferative endometrium but higher than those in early secretory endometrium.“. ‘* Three of the 17 tissues studiedZ7 (about 18 per cent)

had levels as low as those seen in early secretory endometrium. The differentiation of the tumor did not appear to affect E2R levels. In those patients receiving exogenous estrogen, higher levels were tound in the endometrial adenocarcinomas, indicating that estrogens may stimulate the synthesis of E2R. Patients with endometrial carcinoma \\crt‘ studied before and after treatment with oral medrosyprogesterone acetate at a dose of 60 mg. per day administered for two to 10 days. As shown in Table II, four of scvcn patients showed a significant reduction in E:,R level after this treatment. No attempt was made at estrogen priming, and this interesting experiment should bc performed for it could influence the concent]-ation of progesterone receptors and response of the tissut to exogenous progesterone. E2DH. The activity of EBDH in various endometrial tissues is illustrated in Table III. One can set that cnzyme actlvlty in proliferative endometrium is low, whereas there is a marked increase (approximatel? fourteenfold) during the secretor) phase.“’ ‘I‘hat this change can be attributed to progestational activitv ma) be seen in hoth in viva and in vitro studies. .~drninistration of medroxyprogestet-one acetate during the proliferative phase results in a significant incrcasc in the EzDH activity, while in vitro induction of the ~~~/yrne was seen when proliferative endometrium was incubated for two to three days in medium containing progesterone, at concentrations as low as 20 ng. per milliliter.‘” It was possible to inhibit induction of the enzyme by the addition of actimomycin D, c~ordcc~vpiu. puromycin. or cyclohcximide in the medium. Prc&stational agents including norgestrel, rllctlro~\~pt-omedroxyprogesteronc acetate, R5020 gesterone. (Roussel-Uclaf), and norethynodrel also iuciuced cnzyme activity whereas E2, testosterone. and cortisol did not.“” The EZDH activity of postm~nol~;~~~sal, hyperplastic, or adenocarcinomatous endomctrium was found to be as low as that in proliferative cndomrtrium. This result could be anticipated since progcsterone appears to be the necessarv agent for the induction of this enzyme activity. Prior in vitro studies of tisruc steroid dynamics’” showed that E9 is almost exclusively converted to E, and that E, leaves the tissue. Though E, can be converted to Et. the interconversion of rhe t\\o colnpounds in human endometrium favors the oxidative di~~cc.tioll. Therefore, an increase in EzDH lowers thr c.(mccntration of I$ in the tissue. One mechanism by which prvgestins may XI as antiestrogens is by induction of E,DH. E, formctl as a product of EZ metabolism may not have a direct es-

Volume Number

129 7

Table

II. Effects of Provera

Estrogen

Oral Provera (days X mg.)

Patient

Age Cyr.1

A. P.

69

6 x 60

A. B.

69

6 x 60

A. M.

67

7 x 60

E. M.

40

10 x 60

B. H.

48

4 x 60

0. G.

65

2 x 60

A. C.

68

4 x 60

M. U.

66

5 x 60

L. A.

79

7 x 60

C. M.

70

4 x 60

on endometrial Sampling Before After Before After Before After Before After Before After Before After Before After Before After Before After Before After

metabolism

in endometrium

811

adenocarcinoma (nmoleslmg.

Histology

E@H protein

Well-differentiated Secretory change Well-differentiated Destruction df glands Well-differentiated Destruction of glands Well-differentiated, few glands No secretory change Well-differentiated No secretory change Moderately differentiated No secretory change Moderately differentiated Destruction of glands Poorly differentiated No secretory change Poorly differentiated + sarcoma Destruction of glands Poorly differentiated Slight secretory change

t.rogenic effect since no chromatin-bound El receptor complexes were found in human endometrium after incubation of tissue slices with labeled E,.32 The pattern of metabolism of Ez and E1 in endometrial carcinoma in postmenopausal women corresponded to that of normal proliferative endometrium. Ez was preferentially converted to E,, and El, rather than EB, left the tissue. It would seem then that an increase in the enzyme in endometrial carcinoma would produce a decline of the intracellular concentration of Ez. The oral administration of medroxyprogesterone acetate in our studies led to an increase in the level of E2DH in seven of 10 postmenopausal patients with adenocarcinoma, as shown in Table II. The maximum stimulation observed was fourfold, and this is in agreement with the study of Pollow and associates.33 These results indicate that endometrial carcinoma will respond metabolically to progestins in some patients. No attempts were made in our studies to prime the patients with estrogen before treatment with medroxyprogesterone acetate, and further work will be necessary to evaluate the importance of such priming before it can be concluded that some carcinomas are unresponsive to progestational treatment. The activity of E2DH is much lower in myometrium than in endometrium, and in myometrium, in contrast to endometrium, it is not subject to progestational stimulation during the luteal phase. Decidua has low E2DH activity despite the high levels of progesterone circulating during pregnancy. Since decidua originates from stromal elements, its meager l$DH activity is not

Table

E& (jnnoleslmg.

x hr.)

1.5 3.0 1.5 2.0 3.3 3.5 0.9 2.0 1.9 2.0 0.7 1.5 1.4 3.2 1.3 3.5 0.6 0.6 0.9 1.2

DNA)

2.4 2.0 3.7 2.0 4.0 1.6 2.1 0.5 0.8 1 .o 3.1 0.4 1.0 0.7 -

III. EBDH activities

2

N-l Proliferative After progestin administration Secretory Early (days 17-19) Middle (days 20-23)

Late (days 24-28) Hyperplastic (postmenopausal) Adenocarcinoma (postmenopausal) Well-differentiated Exogenous estrogen users Poorly differentiated

20 5

1.4 4.7

0.5 2.0

6 7 5

17 18 I1 1.2

5 5 4 0.6

16 3 3

1.8 1.3 1.2

0.8 0.5 0.4

7

(From Gurpide and associates.*’ Published mission of the Journal of Steroid Biochemzstry.)

with

the

per-

unexpected, for Scublinsky and associate? have demonstrated histochemically that the enzyme is localized in the glandular epithelium of the endometrium.

Comment Within the limits of the parameters studied, that is EzR and EzDH levels, we noted essentially similar values for endometrial carcinoma and endometrial hyperplasia from perimenopausal or postmenopausal subjects and for normal proliferative endometrium from cycling women. They would appear to have a

812

Gurpide, Tseng, and Gusberg

wtntnon

hormonal

absence wth

of

environment

progesterone.

endometrial

those

found

patients

in

failed

characterized

There

were

carcinoma

with

secretorv

endometriutn,

to respond

bv the

some

levels

patients

to progestational

as

sotne

treattnent

with an increase in the E:,DH level or a decrease in the E2R level; as noted above, all of the normal subjects

wsponcled

l+:,K

in

those

in the proliferative

the

carcinoma

reported

appears

above, to

be

.lYhe

specificity

level

of

F.?DH lack

of

phase.

the

specific

for

mitotic

the

second

cot-respond

regulation

in glands

during

peak

tnitotic

of

wave

‘&

negative

f&t from

for

the

the

is obscrvcd

EZDH 0.

G. treated

predictive tins

ant1

in

rion

otE,DH,

thr

results

usefulness

of

presented

here

a short-term

in vivo

tients who are likely to respond gestin.

The

tnust

be

studies cnqtne glands obvious

of

effect

of treatment

evaluated

in

the

in

the

samples. glandular

suggest

to identify

hormone

pa-

to treatment

with pro-

on the activity

of EzDH

conjunction Due

test

the potential

to the epithelium,

with

histologic

localization destruction

of

the of

during treatment. a definite and histologicall) response to progestins, may obscure an inctx-

and

based

offer

in primary

will

be

in viva of the

a method and

OI .tn tzf-

evident in the

bctot-t,

tissue

~lccessar)-

secondary

\virh ;tnd

induc-

appe;tt

tlcfittv the

could

role

pt oges01

It \\~oultl

in individual the

the

of cttclonic~-

or in vitro.

of assaying

Pa-

a( cta~c

to ~'\aluate

changes

tumor

art!

ic’.g., ottc’

to expect that such studies sensitivity

\\;I>

alho .tpp;trcttt 011 tttc. other

treatment

of histologic,

either

rhct-c

ol‘glattds~

twponsiveners

on short-ttwn

evaluation

reasonable

The

Itut

tnedroxvprogestct

of a test for

va1~1e

levels,

activitr.

become

change

days). efforts

ot

enzyme-itttluc tiort tt’st. A. M., in Table II. stto\\c%d no sensitive

tnay with

the

011

inrrrl,t.c-tatiott

(destruction

detectable

glands.

sttt(lic\

on the endotnrtriuttt. in (stromal?) E:?R levels.

induction

only t\z’o (:ollahorative

remaining

erroneous

E,DH

evidence

of progestin the decline

trial cancer

secretor)

plasma

of the

the

biochemical

possible

results

morphologic

in

and

For instance, patient tnc’reasc tn endometrial

of

esplain

activity

histologic prevent

tient

activity

enzymatic

histologically

intracellular the

could

tissue

hand. glands.“’

of

through

luteal

IO

on E,DH

endomctrial

epithelium

activity at

the

the

the

of

“I. *‘. ‘ji

of progesterone

progesterone glandular

even

I\\-hereas

effect

~I‘he levels

generally

workers.“~

the of

E:i by

in

study

by other

‘4s noted

phase.

in

C:ombined

of I;‘lsR as low and

ment

patients

of pt-ogcstins

treatment.

The collaboration of the staff of the Obstetrics and Gynecology, particularly and C:. .L\. Marin. is recognized. Miss provided skillful technical assistance.

Department of Drs. G. Deppc (:vnthia Namit

REFERENCES

Gusherg, S. B.: Precursors of corpus carcinoma estrogen and adenotnatous hyperplasia, AM. J, OBSTET. Gnvwot.. 54: 905, 1947. Gusberg, S. B.: Hormone dependence of endomerrial cancer, Ohstet. Gynecol. 30: 287, 1967. Siiteri, P. K.. and MacDonald, P. C.: The role of. exrraglandular estrogens in human endocrinology, in Handbook of Physiology. sect. 7. part 1, Washington, D. C., 1973, vol. 2, American Physiology Society, p. 615. Rizkallah, T. H., Tovell, H. M. M., and Kelly, W. C.: Production of estrone and fractional conversion of circulating androstenedione to estrone in women Hith endometrial carcinoma, J. Clin. Endocrinol. Metab. 40: 1045. 1975. Hausknecht, R. U., and Gusberg, S. B.: Estrogen metabolism in patients at high risk for endometrial carcinoma. 11. AM. :J. OBSTET. G;NECOL. 116: 98, 1973. Ziel. H. K.. and Finkle. W. D.: Increased risk of endometrial carcinoma among users of conjugated estrogens. N. Engl. J. Med. 293: 1167. 1975. Smith, D. C., Prentice, R., Thompson, D. 1.. and Herman. W. L.: Association of exogenoui estrogen and endometrial carcinoma. N. Erwl. 1. Med. 293: 1164. 1975, Mack. T. M., Pike, M. c”., Henderson, B. E.. Pfeffer. R. I., Gerkins, V. R., Arthur, M., and Brown, S. E.: Estrogens and endometrial cancer in a retirement community, N. Engl. J. Med. 294: 1262. 1976.

Y. Gusherg. S. B.: The individual at high risk f’ot endomcrrial carcinoma, AM. J. OBSTET. Gu~~cor.. 126: 535. 1976. 10. Kelly. R. M.. and Baker, W. H.: The role of progesterone in human endometrial cancer. Cancer Kc5. 25: 1190, 1965. 11. Reifenstrin, E. C.: The treatment of advanced wdotnetrial cancer with hydroxvprogesteronc caproatc. Gvnecol. Oncol. 2: 377. 1974. 12. Twng, L., and Gurpide. E.: Nurlrar con( entr‘ltion 01 estradiol in superfused slices of human endomcatril1m. Asr. J. OBSTET. GYNECOL. 114: YY5, 1972. 13. Tteng, L.. and Gurpidr. E.: Estradiol anti ‘L(tndihydroprogesterone dehydrogenase activities in human endometrium during rhe menstrual cycle, Endoct-inolog\ 94: 419. 1974. 1-k. Brush, M. G.. Taylor, R. W.. and King. R. J. B.: The uptake of [6,7-3H] oestradiol by the normal human reproductive tract, J. Endocrinol. 39: 599. 1967. 15. Robertson, D. M., Mester, J., Beilby, J., Steele, 5. J., and Kellie, A. E.: The measurement of high affinitl ocstradiol rrceptors in human uterine endometrium and myometrium. Acta Endocrinol. 68: 534, 197 1. 16. Limpaphoyom, K., Lee. C.. Jacobson, H. I.. and King, T. H.: Estrogen receptor in human endometrillm during the menstrual cycle and early pregnancy, .4&l. J. OBSTET. (;YNECOL.

17.

Trams,

111:

G.. Engel.

1064,

1971.

B., Lehman.

F.. and Maass,

H.: Specific

Volume Number

18.

19.

20.

21.

22.

23.

24.

25.

26.

129 7

Estrogen

binding of oestradiol in human uterine tissue. Acta Endocrinol. 72: 351, 1973. Evans. L. H.. Martin, G. D.. and Hghnel, R.: Estrogen receptor concentrarion in normal and pathological human uterine tissues. J. Clin. Endocrinol. Metab. 38: 23, lY74. Croaker. S. G., Milton. P. J. D., and King, R.J. B.: Uptake of [6.7-“H] oestradiol-l7P by normal and abnormal human endometrium. J. Endocrinol. 62: 145, 1974. Pollow, Ii., Liibbert, H., Boquoi, E., Kreuzer, G., and Pollow, B.: Characterization and comparison of receptors for 17p estradiol and progesterone in human proliferative endometrium and endometrial carcinoma, Endocrinology 96: 3 19, 1975. Bayard. F.. Damilano. S., Robel, P., and Baulieu, E. E.: Recepteurs de l’oestradiol et de la progesterone dans I’endometre humain us tours du cycle menstruel, Acad. Sci. (1)) (Paris) 281: 1341, 1975. Flickinger, G. L., Elsner, C.. Illingworth, D. V.. Muechler. K. K., and Mikhail, G.: Estrogen and progesterone receprors in the female genital tract of humans and monkeys, .4nn. N. Y. Acad. Sci. 286: 180, 1977. Tseng, L., and Gurpide. E.: Effects of progestins on estradiol receptor levels in human endometrium, J. Clin. Endocrinol. Metab. 41: 402. 1975. Hrenner, R. M.. Resko. J. A., and West, N. B.: Cyclic changes in oviduct morphology and residual cytoplasmic cstl-adiol binding capacity induced by sequential estradiol-progesterone treatment of spayed rhesus monkeys. Endocrinology 95: 1094, 1974. Hsurh. A. .J. W.. Peck, E. J., and Clark, J, H.: Progesterone antagonism of the oestrogen receptor and ~)estl-ogen-inducetl uterine growth, Nature 254: 337, 1975. West. N. B., Vcrhage. H. G., and Brenner, R. M.: Suppression of the estradiol receptor system by progesterone

Discussion DR. JOHN MORRIS, New Haven, Connecticut. In studies on endometrial cancer, Pollow and co-workers, in Berlin, related E;DH activity as well as progesterone-receptor levels to the degree of tumor differentiation. My first question is: Would evaluation of the degree of differentiation of the tumor be as good a guide to identify patients who are likely to respond to progesterone treatment as the receptor and enzyme studies being performed? Ir has also been pointed out by the authors that the endometrial cells rather than the myometrium OT stroma contain the high E2R and E,DH levels. As the percentage of epithelial cells varies considerably from one area of a sample to another. how does one correct for the proportion of stroma or myometrium present? One might ask, “What is an estrogen?” Dr. Arnold Eisenfeld in our Department at Yale has been involved with

studies

of cytosol

receptors

rather

than

nuclear

binding. He and Dr. Amnon Mackler observed that competition with Es for binding sites was directl) porportional to the uterotropic cally estrogenic) activity of the statement which caught my eye

that. “E, formed

as a product

(and thus the biologicompounds tested. A in the manuscript was

of I$ metabolism

may not

27.

28.

29.

30.

31.

32.

33.

34.

35.

metabolism

in endometrium

813

in the oviduct and uterus of the cat, Endocrinology 99: 1010, 1976. Gurpide, E., Gusberg, S. B., and Tseng, L.: Estradiol binding and metabolism in human endometrial hyperplasia and adenocarcinoma, J. Steroid Biochem. 7: 891. 1976. Pollow, K., Liibberr, H., Boquoi, E.. Kreuzer. (;., and Poliow, B.: Characterization and comparison of receptors for 17/3 estradiol and progesterone in human proliferative endometrium and endometrial carcinoma, Endocrinology 96: 319, 1975. Tseng. L., and Gurpide, E.: Induction of human endometrial estradiol dehydrogenase by progestins. Endocrinology 97: 825, 1975. Tseng, L., Gusberg, S. B., and Gurpide, E.: Estradiol receptor and 17/3 dehydrogenase in normal and abnormal human endometrium, Ann. N. Y. Acad. Sci. 286: 190, 1977. Tseng, L., Stolee, A., and Gurpide. E.: Quantitative studies on the uptake and metabolism of estrogens and progesterone by human endometrium, Endocrinology 90: 390, 1972. Tseng. L., and Gurpide. E.: Effect of estrone and progesterone on the nuclear uptake of estradiol hy slices of human endomerrium. Endocrinology 93: 245, 1973. Pollow, K., Boquoi. E., Liibbert, H., and Pollow, B.: Effect of gestagen therapy upon 17p hydroxysteroid dehydrogenase in human endometrium adenocarcinoma. J. Endocrinol. 67: 131, 1975. Scublinsky. A., Marin, C., and Gurpide. E.: Localization of estradiol 17p dehydrogenase in human endometrium, J. Steroid Biochem. 7: 745, 1976. Terenius, L., Lindell, A., and Persson, B. H.: Binding of estradiol 17p to human cancer tissue of the female genital tract. Cancer Res. 31: 1895, 1971.

have a direct estrogenic effect .” In view of the so-called “cstrone hypothesis” put forth by Siitcri and MacDonald, I wonder if Dr. Gurpide would care to comment on this. I have the distinct impression that the authors believe that progestational agents--with Dr. Joseph Zander here we should use the term “gestagens”-are the “good guys” and estrogens are the “bad guys.” Nonetheless, Griffiths and associates observed that the addition of gestagens to estrogen administration in castrated rabbits did not reduce the incidence of endometrial cancer and that the administration of 1%nor steroids actually increased the incidence. While various gestagens are often considewd as a single group, the C-21 compounds, such as progesterone, medrosyprogesterone acetate, chlormadinone, or R5020, differ from most 19-nor compounds, such as norethindrnne or norethynodrel, in that: (1) the 19-nor compounds will not maintain pregnancy in oophorectomized rats or rabbits; (2) they are, like estrogens, uterotropic, although the doses required may be considerably greater; (3) in sufficient doses they will function as postcoital contraceptives; (4) they will not produce the nucleolar channel systems in human endometriutn that the C-2 1 gestagens produce.