ARTHUR L. HÀSKINS, M.D. ERICA F. MOSZKOWSKI, M.D. VICTORIA P. WHITELOCK, M.D.
Baltimore, Maryland.
A clinical and laboratory re-evaluation.
From the Department of Obstetrics and Gynecology, University of Maryland School of Medicine. Presented (by invitation) at the Ninety-first Annual Meeting of the American Gynecological Society, Hot 'Springs, Virginia, May 23-25, 1968.
Abstract
REVIVAL OF interest in estriol has been occasioned by recent observations of variations in urinary. estriol during compromised human pregnancies. In the past, research interest in estriol had been primarily concerned with specific tissue response to the hormone. These studies produced a number of inconsistencies and contradictions concerning estriol and its estrogenicity. The usual assumption that estriol is biologically inert could not be verified, since there were conditions in which estriol was more estro-genic than estradiol or estrone.
Introduction
It is quite apparent that the method by which estrogenicity is evaluated is of the greatest importance. However,. it is difficult to determine which procedures indicate true estrogenicity with greatest significance. Table I indicates some of the variation in the estrogenicity of estriol relative to the other major estrogens as reported by several investigators under ; stipulated testing' conditions. There is excellent agreement, as expressed by Sealey and Sondern,[1] Evans, Varney, and Koch,[2] and Huffman and Groliman,[3] that estriol is less estrogenic than estradiol. Szego[4] indicates to the contrary that estriol is highly estrogenic when compared to estrone and estradiol. The difference in the observations could not be totally related to the test- conditions since, in some instances, the conditions were similar although the findings were at variance.
Table 1.
The effect of the solvent on the estrogenicity of the hormone has also been at issue. As summarized in Table II, Szego, as well as Bum and Elphick,[5] observed an enhancement of the estrogenicity of estriol whenthe solvent was aqueous. Contrarily, Zondek and Sulman[6] reported that an aqueous solvent for estriol reduced the estrogenicity to a 10 per cent level. It is of some interest to note that in addition to technique variation, the “aqueous solvent” varied in constitution.
Table 2. Estrogenicity of estriol in lipoid and “aqueous” solutions.
Discrepancies in the results of estrogenicity studies concerned with the clinical effec-tiveness of estriol in humans were also noted. When, estrogenicity was determined by vaginal cytology, Table III, Brown and Bradbury[7] were unable to demonstrate a positive effect with 1 mg. of estriol daily for 10 days. Mack,[8] however, with, the same dosage schedule demonstrated a positive cytologic effect similar in order to that achieved with estrone, estradiol, and stilbes- trol. Puck[9] observed a positive vaginal cytologic effect with 0.1 mg. estriol daily for 5 days.
In determining estrogenicity of estriol by withdrawal bleeding in women, Table IV, Soule[10] noted this phenomenon in one patient. He compared the effectiveness of estriol as being approximately that of estradiol and stilbestrol. Puck observed that estriol given at 5 mg. a day for 6 days would not induce withdrawal bleeding. In addition to this, there was no evidence of endometrial stimulation by microscopic examination, although there was stimulation of the cervical and vaginal mucosa at this and lower dose levels.
Table 3. Estrogenicity of oral estriol in humans as determined by vaginal cytology.
Table 4. Estrogenicity of estriol in humans as determined by withdrawal bleeding.
One of the more interesting chapters in the estriol story is the concept advanced by Puck, in which he postulates a type of polarity attributable to estriol. This, it is suggested, results in a manifestation of estriol estrogenicity in the lower portions of the human reproductive tract (vagina and cervix) but with little or no effect upon the body of the uterus, or endometrium.
It was this concept, as well as the conflicting reports concerning the estrogenicity of estriol, that stimulated us to review the problem in our laboratories. The estrogenic effect of estriol in the immature mouse as related to uterine weight with both aqueous and lipoid solvents was to be studied. In addition,-the estrogenicity of the hormone № women was to be determined through the evaluation of the maturation index of the vaginal mucosa, ferning of the cervical mucus, estrogen withdrawal bleeding, progesterone withdrawal bleeding, and microscopic study of the endometrium after estriol administration. The estrogenic effects of estriol in women are the subject of the current presentation.
Materials and methods
Estriol, ethinyl estradiol, and stilbestrol were used throughout the study. Each was given orally. The major part of the study was concerned with estriol administration. The, other two substances were administered to small control groups of patients to provide a positive estrogenic control in addition to the negative control in the estriol group.
Women to receive estriol were selected from inpatients and outpatients at Baltimore City Hospitals and University of Maryland Hospital. The patients were selected because of evidence of ovarian failure with amenorrhea and atrophic change in the vaginal mucosa. The patients ranged in age from 47 to 94 years with the average of 66 years. Each patient received 1 mg. of estriol daily by mouth for 28 days.
Vaginal smears for cytologic study were obtained through scraping the lateral vaginal wall with a wood spatula. This procedure was followed in order to provide a standard specimen relatively free from uterine - contamination and from, exfoliated vaginal pool cells. The material obtained was smeared on a glass slide and quickly fixed in 95 per cent alcohol. Subsequently the smears were prepared with Shorr’s stain, the slides were then read by two investigators, and, whenever possible, 100 Cells were read and differentiated into parabasal, intermediate, and superficial cells. The cervical mucus was studied ln 30 patients, 10 of whom received estriol; 10 received 0.1 mg. of ethinyl estradiol daily for 2 - weeks and 10 patients received 0.1 Wg- of stilbestrol daily for 2 weeks. Crystallization of the cervical mucus was demon-strated by microscopic study.
Three patients received a synthetic pro-gestin, megestrol 5 mg. (daily for 5 days) on completion of .the estriol regimen. The purpose of this procedure was to demonstrate the potential of progestin withdrawal bleeding from the estriol-stimulated endometrium.
Two hysterectomy specimens were obtained from patients receiving estriol for 28 days prior to operation. The endometrium was examined for histologic evidence of estrogen stimulation.
Results
Estriol effects on the vaginal epithelium.
A significant change in the cell population of the vaginal epithelium was noted after 2 weeks of estriol administration (Table V). The parabasal cells decreased by approximately 50 per cent. There was a concomitant increase in the intermediate and superficial cells. The changes apparent at 2 weeks of administration were not significantly changed at the 4 weeks’ medication level.
Table 5. Estriol effect on vaginal epithelium maturation index.
Comparison with stilbestrol and ethinyl estradiol is shown in Table VI. Stilbestrol at a dosage level of 0.1 mg. daily appears capable of inducing greater estrogen stimulation of the vaginal mucosa than estriol at the 1 mg. level. Ethinyl estradiol at the same dosage level as stilbestrol shows a greater estrogenic effect than stilbestrol or estriol.
Table 6. The maturation index of the vaginal epithelium after 14 days of therapy.
Cervical mucus. In 10 patients treated with estriol at 1 mg. a day for 28 days, a positive fern test was noted in 2 patients. The remaining were negative. In similar groups of patients treated with ethinyl estradiol and stilbestrol at 0.1 mg. levels, all patients were found to have positive fern tests as indicated in Table VII.
Table 7. Ferning of cervical mucus after 14 days of therapy.
Estrogen withdrawal bleeding. Three patients of the 60 treated with estriol showed evidence of estrogen withdrawal bleeding following therapy. This occurred within a week of the cessation of therapy and consisted of spotting for 2 to 3 days. As indicated in Table VIII, each of the patients manifesting estrogen withdrawal bleeding was noted to have stimulation of ' the vaginal mucosa at a level significantly greater than the mean of the study group.
Table 8. The maturation index of the 3 patients exhibiting estrogen withdrawal bleeding.
Progestin withdrawal bleeding. Three patients were given 5 mg. of megestrol by mouth for 5 days upon the completion of the 28 day course of estriol. As indicated in Table IX, only one patient showed evidence of progestin withdrawal bleeding. This patient showed additional evidence of estro- genicity greater than that observed in the rest of the group in that the fern test was positive and the maturation. index showed an exaggerated estrogen response.
Table 9. Megestrol withdrawal bleeding.
Endometrial stimulation. Hysterectomy was accomplished on 2 patients who were in the estriol study group. Minimal estrogen stimulation of the endometrium was noted. The maturation index at the time of hysterectomy in each, respectively, was 0-84-16 and 14-75-11.
Conclusions
When given orally at a dose level of 1.0 mg. daily for 28 days, estriol produced an estrogenic effect in women. There , is stimulation of the vaginal muCosa which becomes apparent by the second week of administration and remains constant through the following 2 weeks of administration. Return to pretreatment levels of maturation of the vaginal mucosa is..noted on the smears taken 4 weeks after therapy. When compared to the vaginal cytologic effect of stilbestrol and ethinyl estradiol, it is noted that 1 mg. of estriol does not induce as great a degree of estrogenic stimulation of the vaginal mucosa as does 0.1 mg., stilbestrol. Ethinyl estradiol at this dosage level induces an intense estrogenic effect on the vaginal mucosa.
Other evidence of weak estrogenicity of estriol is seen in the occasional development of a positive fern test in the cervical mucus as compared to the 100 per cent development of positive fern tests with smaller amounts of ethinyl estradiol and stilbestrol.
Estrogen withdrawal bleeding with estriol occurred in 3 patients of the 60 study group. Withdrawal bleeding following the admin-istration of a progestational agent occurred in one of three trials.
The studies indicate that estriol is estrogenic in the various modalities tested. The degree of estrogenicity is relatively slight and equivalent to less than 0.1 mg. , of stilbestrol.
Estriol and megestrol were supplied by Organon, Inc., and Mead Johnson & Company, respectively.
References
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Evans, J. S., Varney, R. F., and Koch, F. C.: Endocrinology 28: 747, 1941.
Huffman, M. N., and Grollman, A.: Endocrinology 41: 12, 1947.
Szego, C. M.: Fed. Proc. 9: 124, 1950.
Burn, J. H., and Elphick, G. K.: Quart. J. Pharmacol 5: 192, 1932.
Zondek, B., and Sulman, F.: Endocrinology 49: 417, 1951.
Brown, W. E., and Bradbury, J. T.: J. Clin. Endocrinol. 9. 725, 1949.
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Puck, A.: München, med. Wchnschr. 99: 1 1957.
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