Comparative studies of the ethynyl estrogens used in oral contraceptives III.
Effect on plasma gonadotropins
JOSEPH
W.
GOLDZIEHER,
ARMANDO C.
DE
LA
PENA,
M.D. M.S.
BRANDON
CHENAULT,
M.D.
JANDRO
CERVANTES,
M.D.
ALE
San Antonio,
Texas,
and Mexico,
D. F., Mexico
Twenty-one-day treatment cycles of ethynylestradiol or mestranol at dosages of 50 to 100 pg per day were administered to 191 normal volunteer women for six cycles, followed by six cycles of this estrogen treatment combined with 2.5 mg. of norethindrone acetate, 2 mg. of megestrol acetate, or 0.5 mg. of norgestrel. The drugs were prepared to insure uniform bioavailability. Plasma FSH and LH were determined by radioimmunoasay during the last week of medication intake in each cycle. In another study, a large number of blood samples were obtained at various times during the menstrual cycle from women using IUD’s (as a control population) and from women who had been taking oral contraceptives regularly for 5 to I2 years. With the various estrogen treatments, the median FSH level showed no change at any estrogen dose at the end of the first cycle. From the second cycle on, a stable, dose-related fall was obtained with the 80 or 100 ag per day doses. The addition of any of the three progestins caused a prompt, stable, further fall in FSH level. By contrast, the median LH level rose in the first cycle with all estrogen regimens, and then fell progressively in a dose-related fashion in cycles 2 to 6. The addition of a progestational agent also caused a further prompt and stable fall in LH during cycles 7 to 12. Except for a minimal indication of greater LH suppression by ethynylestradiol as compared to mestranol at 50 pg per day, all other indices and dosages showed ethynylestradiol and mestranol to be essentially equipotent under these experimental conditions. Long-term administration of oral contraceptives produced a comparable degree of gonadotropin suppression. There was a suggestion of slightly less FSH suppression with agents using 50 to 75 fig per day of estrogen than from those with 100 pg per day. Both in normal controls (IUD cycles) and in cycles under chronic treatment with oral contraceptives, pulses of both FSH and LH were seen with some frequency, at times distant from the “periovulatory” period. The significance and origin of these random FSH and LH pulses is unknown.
From the Southwest Foundation for Research Education, San Antonio, and the Asociacion Pro-Salud Maternal, Mexico, D. F., Mexico. This study was supported Agency for International by Wyeth Laboratories, Received Revised Accepted
for
T H E I N T E R A C T I 0 N between estrogen and the hypothalamo-pituitary gonadotropic system has been investigated for decades. The more sophisticated the methodology, the greater are the complexities that appear. The concept of a triphasic dose-response, observed both in experimental animals and human subjects’ serves only as an initial approach to the problem. Alteration of gonadotropin secretion is not only a function of the dose and duration of estrogen treatment, but is also infiuenced by sex, age, presence or absence of gonads, stage of the menstrual cycle in primates, and numerous other factors. The pulsa-
by contract csd/2821, Development, and Radnor, Pennsylvania.
publication
October October
and
August
23, 1974.
3, 1974. 8, 1974.
Reprint requests: Joseph W. Goldzieher, M.D., Director, Clinical Sciences and Reproductive Biology, Southwest Foundation for Research and Education, P.O. Box 28147, 8848 W. Commerce St., San Antonio, Texas 78284.
625
626
Goldzieher
et al.
tile nature of gonadotropin secretion presents still another facet of the problem. Under these circumstances, it is to be expected that clinical investigations of the effect of ethynyl estrogens on the gonadotropic system yield apparently inconsistent results. A variety of dose levels has been examined in postmenopausal or chronically castrated human subjects. At 5 and 10 pg of ethynylestradiol (EE) per day, only minimal decreases in plasma gonadotropins are observed after 2 weeks, and only in about half of the subjects.’ At 20 pg per day plasma FSH levels decrease promptly, eventually falling to about 25 per cent of the control value. There is little effect on plasma LH level, which may decline slowly and rebound after the drug is stopped. At 50 pg per day, both FSH and LH decline at once. With mestranol at the same dose level, the decline may be slower.’ Dose levels of 100 pg per day or more produce about the same result,“. 4 although a continuing fall of FSH for at least 8 days, and unpredictable changes in LH, sometimes with a posttreatment rebound, have been described.” Several investigators’, 1, 5 have commented on the unpredictability of the response to a given dosage, and also on the occasional appearance of LH surges early in the treatment course, even with divided, high-dose regimens. In postpartum women, where the gonadotropic mechanism is in a suppressed state, administration of 15 pg of EE per day produces occasional rebound peaks (of FSH more often than of LH) at the end of 5 days’ treatment; higher doses (30 and 45 pg per day) have less effect.” In normal cycling women, the effect of ethynyl estrogens had already been examined when bioassays specific for urinary FSH and LH were available. Stevens and his associates’, 8 demonstrated that very low doses, such as 10 pg of mestranol per day, elicit substantial increases in urinary FSH, with occasional erratic increases in LH excretion as well. At 50 pg per day or more, FSH excretion is depressed, but LH excretion continues to show erratic increases. Measurements of plasma gonadotropin levels have been carried out at higher doses. Using 80 pg per day of mestranol in a sequential oral contraceptive regimen, Swerdloff and Odell” observed bizarre patterns of LH peaks without concurrent FSH elevations, as well as ablation of the FSH rise which normally occurs early in the cycle. With 100 pg per day of EE given in the first few days of the cycle, Cargille and associatesI reported a drop in FSH and LH levels and a rebound after
termination of the treatment on day 7. This has becx-1 seen by other+l” with doses of 200 to -I-n0 rcg per day given on cycle days 3 to 5, but no LH rrhound was observed, and FSH recovered only graduall\-. When EE at 100 pg per day is given on cycle days 9 to 18,“’ plasma FSH shows a transitory elevation, a decline, and then a rebound ; LH shows a sustained elevation with a mid-cyc.le maximum, a gradual decline, and then a rebound. Longitudinal studies such as these are essential to an understanding of gonadotropin dynamics. However, the investigations are complicated by marked individual variations and by the limited number of subjects available for daily blood sampling. Another, complementary form of information may be gained by cross-sectional studies, which permit sampling of very large numbers of subjects and statistical evaluation of the findings. In a previous cross-sectional study in which normal subjects and individuals on oral or injectable steroid contraceptives were investigated,‘:’ it was possible to identify most of the features of plasma FSH and LH behavior during the normal cycle, except for details of the mid-cycle cannot be pinpointed by a randomsurge, which sampling regimen. Moreover, the qualitative effect of combination and sequential oral contraceptive regimens could be surveyed over periods of many years of use, and these parameters could also be examined during the prolonged use of injected medroxyprogesterone acetate. This study did not permit a comparison of the effect of different doses of steroidal contraceptive agents of uniform bioavailability, nor of different contraceptive formulations. Such data have now been secured in the course of two clinical investigations. The first is a comparative study of ethynylestradiol and mestranol at various dose 1evels.l’. l3 The second examined plasma gonadotropins in women who had engaged in continuous use of oral contraceptives for 5 to 12 years. Subjects using intrauterine devices (IUD) served as one of the control groups. Material
and
methods
The first study examined the response of normal cycling women to 21 day regimens of ethynylestradiol (EE) or mestranol of uniform bioavailability, at various dose 1evels.l’ Between 25 and 40 subjects were enrolled at each dose level. Each regimen was continued for six cycles, after which the same dose of estrogen, now combined with 2.5 mg. of norethindrone acetate, 2 mg. of megestrol acetate, or 0.5 mg. of dl-norgestrel, was continued for another six cycles. About one third of the original group of subjects
Volume Number
122 5
Table
I. Number
Ethynyl
of FSH
or LH
determinations
by cycle, drug,
estrogens
contraceptives.
III
627
100
pg
and dose*
Cycle 1 2 3 4 5
1 Mestmn02, 50 cg I ( Mestranol, 80 cg 1 EE, 50 pg 37 26 25 37 26 24 36 25 22 23 16 27 20 15 26 25 16 23 t” 19 17 27 8 17 17 12 9 13 14 12 10 14 13 16 11 16 13 12 12 16 15 19 Total 200 241 290 *All IUD cycles and control cycles of drug users: 50%. Total: 1,785.
at each estrogen dose level was randomly assigned to one of these combinations. A total of 191 subjects, including 30 IUD controls, were observed for an aggregate of 1,785 cycles. Initial estrogen type and dose assignment was randomized except for subjects receiving 50 Fg of EE or mestranol; this level required the additional precaution of concurrent use of an IUD. Plasma samples were obtained during the last week of drug intake whenever possible; no samples from the periovulatory time of the cycle are included. Two sets of controls were used. The first consisted of blood samples drawn from these subjects at various times during the menstrual cycle prior to the initiation of the estrogen regimen. The second consisted of blood samples obtained at various times of the menstrual cycle of fertile women using intrauterine devices for contraception; these samples were obtained on cycle days 21 to 25, over the period of time that the estrogen study was ongoing. The second study, carried out in collaboration with the Asociacion Pro-Salud Maternal in Mexico City, consisted of obtaining blood samples for hormone assay from women who had been using IUD’s or various combination-type oral contraceptive preparations continuously for periods of 5 to 12 years. Samples were obtained at random times during the treatment cycle. For data analysis, the dosages employed were divided into two categories. The highestrogen-dose classification was limited to a combination agent consisting of 100 pg of mestranol and 1 mg. of ethynodiol diacetate. The various lower-estrogen-dose regimens included 50 pg of EE or 60 to 75 Kg of mestranol combined with 0.5 mg. of norgestrel, 2.5 mg. of lynestrenol, 4 mg. of norethindrone acetate, or 10 mg. of norethindrone. Plasmas FSH and LH were measured in duplicate
in oral
EE,
80
40 33 24 25 21 17 15 19 17 18 16 - 20 265
Fg
1 Mestranol,
33 30 31 26 24 23 20 1% 20 16
281
by double-antibody radioimmunoassay with materials supplied by the National Pituitary Agency.16> I7 LER 907 was used as the reference material for LH assays and 2nd IRP-HMG for the FSH assay. Human plasma pools with low, intermediate, and high levels were used as additional controls; doseresponse curves were constructed with the use of logit transformation, and values in milli-International Units per milliliter read directly. Quality control was carried out as described by Rodbard and associates.18 Statistical analysis of the data presented some interesting problems. The distribution of the values of plasma FSH and LH appeared to be symmetrical but extremely flat as compared to a Gaussian distribution. Because of the relatively large number of values at both ends of these distributions, sample sizes of 30 or less tend to produce U-shaped distrrbutlons with large discrepancies between the mean and median values. Under these conditions, the median value is considered to be better for representing the central tendency of a particular sample. The samples were tested with the nonparametric Kruskal-Wallis one-way analysis of variance. This was considered to be a more efficient procedure than the median test because it utilized more of the information by transforming the data into ranks, rather than a simple dichotomy of data above and below the median value. Differences between the two estrogens at various dose levels and changes by cycle for each regimen were examined by a Friedman two-way analysis of variance. Results
I. Comparative estrogen study. The data base of plasma gonadotropin determinations is shown in Table I.
i3
=
‘L,
a..*
\, **.\ “3
“P .:.
\D /,d! *.... / b d A. b
UI-
N-
-_
‘. -.
.“..
. ..._., 0
4..” ._. .
$j
....
,...” ‘..,
,_....‘.
. .
‘. -
...’.rj ,..’...’ a::,
?--$” , ’
‘P
,...”
Volume Number
122 5
Ethynyl
0’A“;‘I
0B levels.
CONTROL
DATA.
contraceptives.
III
629
CYCLE
OI’
The plasma FSH values in the control population of IUD users were examined by cycle, and no differences between cycles were observed ; the data were therefore combined into a single group, with a median value of 7.5 m1.U. per milliliter. Next, the pretreatment-cycle data for each of the groups assigned to the various estrogen regimens were compared to each other and to the IUD data by means of the Kruskal-Wallis multi-sample test. The sampled populations were identical, as hypothesized, and the entire set of data was combined and used as a control. B. ESTROGEN ADMINISTRATION. The median values for FSH are shown, by cycle, for each of the treatment regimens (EE 50 and 80 pg per day; mestranol A.
in oral
:‘: 81I :
Fig. 2. Median values for plasma LH levels therapy for six cycles, followed by six cycles to the re,Gmen. A, Administration of mestranol of ethynylestradiol at 50 and 80 cg per day.
FSH
estrogens
CYCLE in groups of subjects receiving cyclic estrogen during which a synthetic progestin was added at 50, 80, and 100 pg per day; B, Administration
50, 80 and 100 pg per day) in Fig. 1. The control data were then compared to each treatment group for the first six estrogen-treatment cycles. In the first cycle, none of the median values for the treatment groups was significantly different from the control level. In the second cycle, 50 pg of mestranol still did not differ from control and 50 pg of EE showed a drop of equivocal significance (0.05 > P > 0.02). The fall in the median values of all the 80 and 100 rg doses was highly significant (P < 0.005). In the succeeding four cycles, 50 pug of mestranol never fell significantly below the control levels, 50 pg of EE showed an equivocal fall at best, and the three higher dose levels maintained significant suppression as compared to the control FSH level. For a further analysis, the control group was
60
4 5, 2 a
LEVELS OF PLASMA FSH by menstrual cycle day in fertile women using an IUD
30
25
0
b
lb Fig.
3A.
Levels
of plasma
3'0
2b CYCLE
FSH
by menstrual
0
LIMIT o OF DETECTIBILITY
>32
DAY
cycle
day
in fertile
women
using
an
IUD.
Volume Number
122 5
Ethynyl
estrogens
in oral
contraceptives.
36'
LEVELS OF PLASMA LH by menstrual in fertile women using an IUD
cycle day
0
0
0
0
0 D D
0
00 0
0
0 0
0
0
0
LIMIT OF DETECTpTY
0 0 0
0
0
0
7 CYCLE
Fig.
3B. Levels
of plasma
LH
by menstrual
cycle
>32
DAY
day
in fertile
women
using
an IUD.
III
631
6 22
Goidzieiier
et ai.
LEVELS OF PLASMA FSH in fertile women using ,015 mg ethynylestradiol or 0.06 - 0.075 or 10 mg norethindrone
Eugynon
& Ovral
Anovlar
A
mg mestranol
and
for 5 to 12 yrs. by
Ortho-Novum
0 0
10mg
0
;
Lindiol
co
A
d
CYCLE DAY Fig. 4A. Levels of plasma FSH in fertile women using 0.05 mg. of ethynylestradiol or 0.06 to 0.075 mg. of mestranol and 0.5 mg. of norgestrel or 2.5 mg. of lynestrenol or 4 mg. of norethindrone acetate or 10 mg. of norethindrone for 5 to 12 years, by menstrual cycle day.
omitted, and a two-way Friedman analysis of variance was performed to examine the differences between dose levels and the changes from cycle to cycle, for each estrogen. Taking only cycles 2 to 6 into consideration, the median FSH values for both EE and mestranol were stable, for each dose level, from cycle to cycle (i.e., a plateau was reached by cycle 2). The increasing dosage of mestranol resulted in progressively lower median FSH values: 7.0, 3.9, and 2.6 m1.U. per milliliter, respectively, for the 50, 80, and 100 pg levels. These differences are statistically significant. The data for EE were similar: 5.8 and 4.2 m1.U. per milliliter, respectively, for the 50 and 80 pg doses, a significant difference. A comparison of EE with mestranol, at each dose level, was also carried out by means of a two-way analysis of variance. There was no significant difference between the two compounds at either the 50 or the 80 pg per day dose levels. Thus, in terms of inhibition of pituitary FSH release, the two estro-
gens appear to have equivalent potency subjects, at least over this dosage range. C.
COMBINED
ESTROGEN-PROGESTIN
in human ADMINISTRA-
For the second half of the study protocol, each of the groups using a particular estrogen regimen was divided into three approximately equal subgroups. The three subgroups continued for six additional cycles to use the same type and dose of estrogen but received, in addition, one of the three progestins concurrently. Statistical analysis of the three progestin subgroups yielded no significant difference in any instance, so the data were combined for each estrogen regimen, as shown in Fig. 1. In evaluating the effect of additional progestin, the median FSH level for cycle 6 of the particular estrogen regimen served as the appropriate control. A highly significant fall in median FSH value (P < 0.005) occurred immediately with the 50 and 80 pg mestranol-progestin combinations. The fall with the 50 pg EE-progestin combination was also significant TION.
Volume Number
122 5
Ethynyl
estrogens
in oral
contraceptives.
III
633
456' 32.4'
276'
>
15 i
LEVELS OF PLASMA LH in fertile women using 0.05 mg ethynylcstradiol or 0.06 -0.075 mg mestnnol and 0.5 mg norgertrol or 2.5 mg lynestrenol or 4 mg norethindrone acetate or 10 mg norethindrone for 5 to 12 yrs. by menstrual cycle day.
Eugynon & Ovral Anovlar
Fig. 4B. Levels
A
0
Ortho
Novum
Lindiol
of plasma LH in fertile women using 0.05 mg. of ethynylestradiol mg. of mestranol and 0.5 mg. of norgestrel or 2.5 mg. of lynestrenol or 4 mg. acetate or 10 mg. of norethindrone for 5 to 12 years, by menstrual cycle day.
at 0.05 > P > 0.02 except for the last cycle. With the 80 pg of EE and 100 pg of mestranol combinations a further decrease in the already low FSH level was not observed. Further suppression either was not feasible or its detection was beyond the sensitivity of the radioimmunoassay. Analysis of variance was applied, by dose and by cycle, to the data of cycles 7 to 12. For each of the five groups, the median FSH level was stable throughout the six successive cycles. No difference in the median FSH values (i.e., the degree of suppression) could be detected in comparing the three mestranol dose levels with each other, nor in comparing the two EE dose levels with each other. Finally, comparison of mestranol with EE combinations at the 50 or 80 pg estrogen level also showed no significant differences. It therefore appears that all these estrogen-progestin combinations show the maximum degree of FSH suppression at the lowest estrogen level tested. These results are consistent
10 mg 0
0
or 0.06 to 0.075 of norethindrone
with data based on ovulation inhibitionI where far lower estrogen dosage in combination with various progestins proved to be as effective as higher doses. LH levels. A. CONTROL DATA. The median LH values of the IUD group showed an unexplained variation during the first six cycles. These data were therefore excluded, and the LH va.lues of the pretreatment cycles of each group assigned to a particular estrogen regimen served as its own control. However, this greatly reduced the sample size of the controls and limited the comparisons which could be made with the treatment values. In any event, they were consistent with the two-way analysis of variance, which has a greater analytical power under these circumstances. B. ESTROGEN ADMINISTRATION (FIG. 2). Compared to its own pretreatment level, each of the mestranol and EE regimens showed a higher median LH value in cycle 1. From then on, there was a highly signifi-
Table Il. Frequency distribution contraceptives for 5 to 12 years
of FSH and LH values in women using high- or low-estrogen (samples taken randomly throughout the cycle)
samples at various FSH levels < 1.2 rnl.U./ml. / > 1.2 < 10.0 mI.U./ml. / > 10 mI.lJ./ml. 30 100 25 28 114 10 Number of samples at various LH levels Number
Estrogen
level
~
Low-dose High-dose I Estrogen
level
Low-dose High-dose
Q
0.4
mI.lJ./ml.
of
/
>
0.4
5.0
mI.U./ml.
5 8
oral
Total A’o. samples
of
155 1.5” Total No. samples
of
125 132
subjects. Particularly interesting in Figs. 3A and 38 are the pulses of FSH and LH (not necessarily concurrent) which are unassociated with the expected time of ovulation. There is an intimation of this phenomenon in some published longitudinal studies of normal cycles, but without any estimate of its frequency. From a practical point of view, such information is important, since diagnostic studies including a single high plasma gonadotropin value (at a nonovulatory time) might be misinterpreted if the normal occurrence of isolated gonadotropin pulses is not recognized. Plasma gonadotropin levels in long-term oral contraceptive users have not been investigated to any extent, although post-treatment return of ovulatory activity (implying normal gonadotropin dynamics) has been extensively documented.lg The plasma FSH and LH levels obtained at random times during treatment cycles of women who had been steroidal contraceptive users for 5 to 12 years are displayed in Figs. 4A and +B. Due to the relatively small number of samples, the data were grouped into low, intermediate, and high FSH and LH levels, and analyzed by the chi-square method. The data are subdivided into those from low-estrogen (50 pg of EE or 60 to 75 pg of mestranol) and higher estrogen (100 pg of mestranol) users (Table II). The frequency distribution of FSH values between low- and high-estrogen users differs only with respect to values of 10 m1.U. or above. More of these values were seen in the low-estrogen group. This difference is significant at P < 0.05. The higher values in the low-estrogen users are randomly distributed throughout the treatment cycle and occur with all the various formulations. The small number of samples does not permit analysis of the influence of different progestins. No significant difference was seen in the distribution of the two sets of LH values.
Volumr
122
Sumbrr
5
Comment These studies with oral EE and mestranol of controlled bioavailability” reveal novel effects on gonadotropin dynamics during 21 day cycles of administration, recognizing that only changes in the last week of each treatment cycle are being measured. The first cycle of estrogen exposure produced no significant change of the median FSH value at any dose of either estrogen. In the second cycle, a dose-related suppression appeared, and remained stable at each dose level for the succeeding four cycles. However, no significant suppression occurred at any time with 50 ,ug of mestranol, and the response to 50 pg of EE was equivocal. Mestranol and EE appeared to be equipotent over the 50 to 80 pg dosage range. The addition of any one of three synthetic progestins to the cyclic estrogen dosage produced a further prompt and consistent suppression of plasma FSH. No qualitative difference could be detected between the progestins at this profound level of suppression nor was there any difference between the various estrogen dosages. The effects on median LH values were quite different, for they rose by the latter part of the first treatment cycle at all dose levels, and then over the next five cycles they underwent a continuous decline which was related to the magnitude of the estrogen dose. EE appeared to be slightly more potent than mestranol at the 50 pg per day level, but at 80 pg per day no difference could be detected. The addition of progestins to the cyclic estrogen treatment produced a substantial further depression of median LH values, just as was seen with FSH, but in this case the level of mestranol seemed to make some difference in the degree of LH suppression. These findings differ from previous observations of the effect of ethynyl estrogens in castrate, postmenopausal, or normal cycling women. The dosage regimens and the time of measurement are quite different, and it is not possible to explain or reconcile these differences in the present state of our knowledge of estrogen-related gonadotropin dynamics; however, the findings give some insight into the relative
Ethynyl
estrogens
in oral
contraceptives.
III
635
potency of the two ethynyl estrogens and the magnitude of their effect in the range commonly used in contraceptive agents. The synergistic effect of the progestins, implicit in the high antiovulatory effectiveness of very low-dose estrogen-progestin combinations, is confirmed by these studies of concomitant gonadotropin levels. The randomly timed samples taken in long-term users of combination oral contraceptives show comparable or perhaps slightly greater degrees of gonadotropin suppression, with a suggestion of less FSH inhibition with the lower estrogen dose agents. What is more interesting, however, is the occurrence of relatively frequent pulses of gonadotropin release. This finding provides further evidence, if any is needed, that the pituitary is not suppressed by these agents in the same sense that the thyroid or the adrenal cortex are suppressed by chronically administered thyroxine or cortisone with resultant cellular involution and even atrophy. Cyclic estrogen-progestin therapy seems, rather, barely to keep gonadotropin release in check, preventing primarily the ovulatory surge (in a very high percentage of cycles) and reducing the general level of gonadotropin most of the time, while permitting occasional surges of gonadotropin output. This phenomenon, currently unexplained, is also seen in the normal cycles of IUD users, and appears to occur with some frequency. In the context of our related studies on the relative potency of ethynyl estradiol and mestranol,llq I5 these results confirm the approximate equipotency of these two compounds, in contrast to most investigations in other nonprimate species.
We wish to acknowledge the participation of Dr. Louis E. Moses, who was clinical supervisor of this project prior to his untimely death, the collaboration of Drs. Edris Rice-Wray and Jaime Gorodovsky of the Asociacion Pro-Salud Maternal, Mexico City, the extensive statistical assistance of Mr. Tazewell Dozier, the technical assistance of Mss. Joyce Mandel and Donna Shannon, and a generous gift of mestranol from Syntex Laboratories.
REFERENCES
1. Hagino, N., and Goldzieher, J. W.: Contraception 1: 131, 1970. 2. Wise, A. J., Gross, M. A., and Schalch, D. S.: J. Lab. Clin. Med. 81: 28, 1973. 3. Franchimont, P., Legros, J. J., and Meurice, J.: Harm. Metab. Res. 4: 288, 1972.
4. Yen, S. S., and Tsai, C. C.: J. Clin. Endocrinol. 33: 882, 1971. 5. Wallach, E. E., Root, A. W., and Garcia, C-R.: J. Clin. Endocrinol. 31: 376, 1970. 6. Crystle, C. D., Sawaya, G. A., and Stevens, V. C.: AM. J. OBSTET. GYNECOL. 116: 616, 1973.
7. 8. 9. 10. Il. 12.
13.
Stevens, V. C., and Vorys, N.: Obstet. Gynecol. Sum. 22: 781, 1967. Stevens, V. C.. Goldzieher, J. W., and Vorys, N.: AM. J. OBSTET. GYNECOL. 102: 95, 1968. Swerdloff, R. S., and Odell, W. D.: J. Clin. Endocrinol. 29: 157, 1969. Cargille, C. M., Vaitukaitis, J. L., Bermudez, J. A., and Ross, G. T.: J. Clin. Endocrinol. 36: 87, 1973. Tsai, C. C., and Yen, S. S.: J. Clin. Endocrinol. 33: 917, 1971. Vaitukaitis, J. L., Bermudez, J. A., Cargille, C. M., Lipsett, M. B., and Ross, G. T.: J. Clin. Endocrinol. 32: 503, 1971. Goldzieher, J. W., Kleber, J. W., Moses, L. E., and Rathmacher, R. P.: Contraception 2: 225, 1970.
14.
15.
16. 17. 18. 19.
Goldzieher, J. W., Maqueo, M., Chenault, C. B., and Woutersz, T. B.: AM. J. OBSTET. GYNECOL. 122: 615, 1975. Goldzieher, J. W., de la Pefia, A., Chenault. C. I~.> and Woutersz. T. B.: AM. J. OBSTET. GYNECOL. 122: 619, 1975. Odell, W. D., Ross, G. T., and Rayford, P. L.: J. Lab. Clin. Med. 70: 973, 1967. Midgley, A. R.: J. Clin. Endocrinol. 27: 295, 1967. Rodbard, D., Rayford, P. L., Cooper, J. A., and Ross, G. T.: J. Clin. Endocrinol. Metab. 28: 1412. 1968. Rice-Wray, E., Correu, S., Gorodovsky, J., Esquivel, J., and Goldzieher, J. W.: Fertil. Steril. 18: 212, 1967.
Effect on plasma gonadotropins
JOSEPH
W.
GOLDZIEHER,
ARMANDO C.
DE
LA
PENA,
M.D. M.S.
BRANDON
CHENAULT,
M.D.
JANDRO
CERVANTES,
M.D.
ALE
San Antonio,
Texas,
and Mexico,
D. F., Mexico
Twenty-one-day treatment cycles of ethynylestradiol or mestranol at dosages of 50 to 100 pg per day were administered to 191 normal volunteer women for six cycles, followed by six cycles of this estrogen treatment combined with 2.5 mg. of norethindrone acetate, 2 mg. of megestrol acetate, or 0.5 mg. of norgestrel. The drugs were prepared to insure uniform bioavailability. Plasma FSH and LH were determined by radioimmunoasay during the last week of medication intake in each cycle. In another study, a large number of blood samples were obtained at various times during the menstrual cycle from women using IUD’s (as a control population) and from women who had been taking oral contraceptives regularly for 5 to I2 years. With the various estrogen treatments, the median FSH level showed no change at any estrogen dose at the end of the first cycle. From the second cycle on, a stable, dose-related fall was obtained with the 80 or 100 ag per day doses. The addition of any of the three progestins caused a prompt, stable, further fall in FSH level. By contrast, the median LH level rose in the first cycle with all estrogen regimens, and then fell progressively in a dose-related fashion in cycles 2 to 6. The addition of a progestational agent also caused a further prompt and stable fall in LH during cycles 7 to 12. Except for a minimal indication of greater LH suppression by ethynylestradiol as compared to mestranol at 50 pg per day, all other indices and dosages showed ethynylestradiol and mestranol to be essentially equipotent under these experimental conditions. Long-term administration of oral contraceptives produced a comparable degree of gonadotropin suppression. There was a suggestion of slightly less FSH suppression with agents using 50 to 75 fig per day of estrogen than from those with 100 pg per day. Both in normal controls (IUD cycles) and in cycles under chronic treatment with oral contraceptives, pulses of both FSH and LH were seen with some frequency, at times distant from the “periovulatory” period. The significance and origin of these random FSH and LH pulses is unknown.
From the Southwest Foundation for Research Education, San Antonio, and the Asociacion Pro-Salud Maternal, Mexico, D. F., Mexico. This study was supported Agency for International by Wyeth Laboratories, Received Revised Accepted
for
T H E I N T E R A C T I 0 N between estrogen and the hypothalamo-pituitary gonadotropic system has been investigated for decades. The more sophisticated the methodology, the greater are the complexities that appear. The concept of a triphasic dose-response, observed both in experimental animals and human subjects’ serves only as an initial approach to the problem. Alteration of gonadotropin secretion is not only a function of the dose and duration of estrogen treatment, but is also infiuenced by sex, age, presence or absence of gonads, stage of the menstrual cycle in primates, and numerous other factors. The pulsa-
by contract csd/2821, Development, and Radnor, Pennsylvania.
publication
October October
and
August
23, 1974.
3, 1974. 8, 1974.
Reprint requests: Joseph W. Goldzieher, M.D., Director, Clinical Sciences and Reproductive Biology, Southwest Foundation for Research and Education, P.O. Box 28147, 8848 W. Commerce St., San Antonio, Texas 78284.
625
626
Goldzieher
et al.
tile nature of gonadotropin secretion presents still another facet of the problem. Under these circumstances, it is to be expected that clinical investigations of the effect of ethynyl estrogens on the gonadotropic system yield apparently inconsistent results. A variety of dose levels has been examined in postmenopausal or chronically castrated human subjects. At 5 and 10 pg of ethynylestradiol (EE) per day, only minimal decreases in plasma gonadotropins are observed after 2 weeks, and only in about half of the subjects.’ At 20 pg per day plasma FSH levels decrease promptly, eventually falling to about 25 per cent of the control value. There is little effect on plasma LH level, which may decline slowly and rebound after the drug is stopped. At 50 pg per day, both FSH and LH decline at once. With mestranol at the same dose level, the decline may be slower.’ Dose levels of 100 pg per day or more produce about the same result,“. 4 although a continuing fall of FSH for at least 8 days, and unpredictable changes in LH, sometimes with a posttreatment rebound, have been described.” Several investigators’, 1, 5 have commented on the unpredictability of the response to a given dosage, and also on the occasional appearance of LH surges early in the treatment course, even with divided, high-dose regimens. In postpartum women, where the gonadotropic mechanism is in a suppressed state, administration of 15 pg of EE per day produces occasional rebound peaks (of FSH more often than of LH) at the end of 5 days’ treatment; higher doses (30 and 45 pg per day) have less effect.” In normal cycling women, the effect of ethynyl estrogens had already been examined when bioassays specific for urinary FSH and LH were available. Stevens and his associates’, 8 demonstrated that very low doses, such as 10 pg of mestranol per day, elicit substantial increases in urinary FSH, with occasional erratic increases in LH excretion as well. At 50 pg per day or more, FSH excretion is depressed, but LH excretion continues to show erratic increases. Measurements of plasma gonadotropin levels have been carried out at higher doses. Using 80 pg per day of mestranol in a sequential oral contraceptive regimen, Swerdloff and Odell” observed bizarre patterns of LH peaks without concurrent FSH elevations, as well as ablation of the FSH rise which normally occurs early in the cycle. With 100 pg per day of EE given in the first few days of the cycle, Cargille and associatesI reported a drop in FSH and LH levels and a rebound after
termination of the treatment on day 7. This has becx-1 seen by other+l” with doses of 200 to -I-n0 rcg per day given on cycle days 3 to 5, but no LH rrhound was observed, and FSH recovered only graduall\-. When EE at 100 pg per day is given on cycle days 9 to 18,“’ plasma FSH shows a transitory elevation, a decline, and then a rebound ; LH shows a sustained elevation with a mid-cyc.le maximum, a gradual decline, and then a rebound. Longitudinal studies such as these are essential to an understanding of gonadotropin dynamics. However, the investigations are complicated by marked individual variations and by the limited number of subjects available for daily blood sampling. Another, complementary form of information may be gained by cross-sectional studies, which permit sampling of very large numbers of subjects and statistical evaluation of the findings. In a previous cross-sectional study in which normal subjects and individuals on oral or injectable steroid contraceptives were investigated,‘:’ it was possible to identify most of the features of plasma FSH and LH behavior during the normal cycle, except for details of the mid-cycle cannot be pinpointed by a randomsurge, which sampling regimen. Moreover, the qualitative effect of combination and sequential oral contraceptive regimens could be surveyed over periods of many years of use, and these parameters could also be examined during the prolonged use of injected medroxyprogesterone acetate. This study did not permit a comparison of the effect of different doses of steroidal contraceptive agents of uniform bioavailability, nor of different contraceptive formulations. Such data have now been secured in the course of two clinical investigations. The first is a comparative study of ethynylestradiol and mestranol at various dose 1evels.l’. l3 The second examined plasma gonadotropins in women who had engaged in continuous use of oral contraceptives for 5 to 12 years. Subjects using intrauterine devices (IUD) served as one of the control groups. Material
and
methods
The first study examined the response of normal cycling women to 21 day regimens of ethynylestradiol (EE) or mestranol of uniform bioavailability, at various dose 1evels.l’ Between 25 and 40 subjects were enrolled at each dose level. Each regimen was continued for six cycles, after which the same dose of estrogen, now combined with 2.5 mg. of norethindrone acetate, 2 mg. of megestrol acetate, or 0.5 mg. of dl-norgestrel, was continued for another six cycles. About one third of the original group of subjects
Volume Number
122 5
Table
I. Number
Ethynyl
of FSH
or LH
determinations
by cycle, drug,
estrogens
contraceptives.
III
627
100
pg
and dose*
Cycle 1 2 3 4 5
1 Mestmn02, 50 cg I ( Mestranol, 80 cg 1 EE, 50 pg 37 26 25 37 26 24 36 25 22 23 16 27 20 15 26 25 16 23 t” 19 17 27 8 17 17 12 9 13 14 12 10 14 13 16 11 16 13 12 12 16 15 19 Total 200 241 290 *All IUD cycles and control cycles of drug users: 50%. Total: 1,785.
at each estrogen dose level was randomly assigned to one of these combinations. A total of 191 subjects, including 30 IUD controls, were observed for an aggregate of 1,785 cycles. Initial estrogen type and dose assignment was randomized except for subjects receiving 50 Fg of EE or mestranol; this level required the additional precaution of concurrent use of an IUD. Plasma samples were obtained during the last week of drug intake whenever possible; no samples from the periovulatory time of the cycle are included. Two sets of controls were used. The first consisted of blood samples drawn from these subjects at various times during the menstrual cycle prior to the initiation of the estrogen regimen. The second consisted of blood samples obtained at various times of the menstrual cycle of fertile women using intrauterine devices for contraception; these samples were obtained on cycle days 21 to 25, over the period of time that the estrogen study was ongoing. The second study, carried out in collaboration with the Asociacion Pro-Salud Maternal in Mexico City, consisted of obtaining blood samples for hormone assay from women who had been using IUD’s or various combination-type oral contraceptive preparations continuously for periods of 5 to 12 years. Samples were obtained at random times during the treatment cycle. For data analysis, the dosages employed were divided into two categories. The highestrogen-dose classification was limited to a combination agent consisting of 100 pg of mestranol and 1 mg. of ethynodiol diacetate. The various lower-estrogen-dose regimens included 50 pg of EE or 60 to 75 Kg of mestranol combined with 0.5 mg. of norgestrel, 2.5 mg. of lynestrenol, 4 mg. of norethindrone acetate, or 10 mg. of norethindrone. Plasmas FSH and LH were measured in duplicate
in oral
EE,
80
40 33 24 25 21 17 15 19 17 18 16 - 20 265
Fg
1 Mestranol,
33 30 31 26 24 23 20 1% 20 16
281
by double-antibody radioimmunoassay with materials supplied by the National Pituitary Agency.16> I7 LER 907 was used as the reference material for LH assays and 2nd IRP-HMG for the FSH assay. Human plasma pools with low, intermediate, and high levels were used as additional controls; doseresponse curves were constructed with the use of logit transformation, and values in milli-International Units per milliliter read directly. Quality control was carried out as described by Rodbard and associates.18 Statistical analysis of the data presented some interesting problems. The distribution of the values of plasma FSH and LH appeared to be symmetrical but extremely flat as compared to a Gaussian distribution. Because of the relatively large number of values at both ends of these distributions, sample sizes of 30 or less tend to produce U-shaped distrrbutlons with large discrepancies between the mean and median values. Under these conditions, the median value is considered to be better for representing the central tendency of a particular sample. The samples were tested with the nonparametric Kruskal-Wallis one-way analysis of variance. This was considered to be a more efficient procedure than the median test because it utilized more of the information by transforming the data into ranks, rather than a simple dichotomy of data above and below the median value. Differences between the two estrogens at various dose levels and changes by cycle for each regimen were examined by a Friedman two-way analysis of variance. Results
I. Comparative estrogen study. The data base of plasma gonadotropin determinations is shown in Table I.
i3
=
‘L,
a..*
\, **.\ “3
“P .:.
\D /,d! *.... / b d A. b
UI-
N-
-_
‘. -.
.“..
. ..._., 0
4..” ._. .
$j
....
,...” ‘..,
,_....‘.
. .
‘. -
...’.rj ,..’...’ a::,
?--$” , ’
‘P
,...”
Volume Number
122 5
Ethynyl
0’A“;‘I
0B levels.
CONTROL
DATA.
contraceptives.
III
629
CYCLE
OI’
The plasma FSH values in the control population of IUD users were examined by cycle, and no differences between cycles were observed ; the data were therefore combined into a single group, with a median value of 7.5 m1.U. per milliliter. Next, the pretreatment-cycle data for each of the groups assigned to the various estrogen regimens were compared to each other and to the IUD data by means of the Kruskal-Wallis multi-sample test. The sampled populations were identical, as hypothesized, and the entire set of data was combined and used as a control. B. ESTROGEN ADMINISTRATION. The median values for FSH are shown, by cycle, for each of the treatment regimens (EE 50 and 80 pg per day; mestranol A.
in oral
:‘: 81I :
Fig. 2. Median values for plasma LH levels therapy for six cycles, followed by six cycles to the re,Gmen. A, Administration of mestranol of ethynylestradiol at 50 and 80 cg per day.
FSH
estrogens
CYCLE in groups of subjects receiving cyclic estrogen during which a synthetic progestin was added at 50, 80, and 100 pg per day; B, Administration
50, 80 and 100 pg per day) in Fig. 1. The control data were then compared to each treatment group for the first six estrogen-treatment cycles. In the first cycle, none of the median values for the treatment groups was significantly different from the control level. In the second cycle, 50 pg of mestranol still did not differ from control and 50 pg of EE showed a drop of equivocal significance (0.05 > P > 0.02). The fall in the median values of all the 80 and 100 rg doses was highly significant (P < 0.005). In the succeeding four cycles, 50 pug of mestranol never fell significantly below the control levels, 50 pg of EE showed an equivocal fall at best, and the three higher dose levels maintained significant suppression as compared to the control FSH level. For a further analysis, the control group was
60
4 5, 2 a
LEVELS OF PLASMA FSH by menstrual cycle day in fertile women using an IUD
30
25
0
b
lb Fig.
3A.
Levels
of plasma
3'0
2b CYCLE
FSH
by menstrual
0
LIMIT o OF DETECTIBILITY
>32
DAY
cycle
day
in fertile
women
using
an
IUD.
Volume Number
122 5
Ethynyl
estrogens
in oral
contraceptives.
36'
LEVELS OF PLASMA LH by menstrual in fertile women using an IUD
cycle day
0
0
0
0
0 D D
0
00 0
0
0 0
0
0
0
LIMIT OF DETECTpTY
0 0 0
0
0
0
7 CYCLE
Fig.
3B. Levels
of plasma
LH
by menstrual
cycle
>32
DAY
day
in fertile
women
using
an IUD.
III
631
6 22
Goidzieiier
et ai.
LEVELS OF PLASMA FSH in fertile women using ,015 mg ethynylestradiol or 0.06 - 0.075 or 10 mg norethindrone
Eugynon
& Ovral
Anovlar
A
mg mestranol
and
for 5 to 12 yrs. by
Ortho-Novum
0 0
10mg
0
;
Lindiol
co
A
d
CYCLE DAY Fig. 4A. Levels of plasma FSH in fertile women using 0.05 mg. of ethynylestradiol or 0.06 to 0.075 mg. of mestranol and 0.5 mg. of norgestrel or 2.5 mg. of lynestrenol or 4 mg. of norethindrone acetate or 10 mg. of norethindrone for 5 to 12 years, by menstrual cycle day.
omitted, and a two-way Friedman analysis of variance was performed to examine the differences between dose levels and the changes from cycle to cycle, for each estrogen. Taking only cycles 2 to 6 into consideration, the median FSH values for both EE and mestranol were stable, for each dose level, from cycle to cycle (i.e., a plateau was reached by cycle 2). The increasing dosage of mestranol resulted in progressively lower median FSH values: 7.0, 3.9, and 2.6 m1.U. per milliliter, respectively, for the 50, 80, and 100 pg levels. These differences are statistically significant. The data for EE were similar: 5.8 and 4.2 m1.U. per milliliter, respectively, for the 50 and 80 pg doses, a significant difference. A comparison of EE with mestranol, at each dose level, was also carried out by means of a two-way analysis of variance. There was no significant difference between the two compounds at either the 50 or the 80 pg per day dose levels. Thus, in terms of inhibition of pituitary FSH release, the two estro-
gens appear to have equivalent potency subjects, at least over this dosage range. C.
COMBINED
ESTROGEN-PROGESTIN
in human ADMINISTRA-
For the second half of the study protocol, each of the groups using a particular estrogen regimen was divided into three approximately equal subgroups. The three subgroups continued for six additional cycles to use the same type and dose of estrogen but received, in addition, one of the three progestins concurrently. Statistical analysis of the three progestin subgroups yielded no significant difference in any instance, so the data were combined for each estrogen regimen, as shown in Fig. 1. In evaluating the effect of additional progestin, the median FSH level for cycle 6 of the particular estrogen regimen served as the appropriate control. A highly significant fall in median FSH value (P < 0.005) occurred immediately with the 50 and 80 pg mestranol-progestin combinations. The fall with the 50 pg EE-progestin combination was also significant TION.
Volume Number
122 5
Ethynyl
estrogens
in oral
contraceptives.
III
633
456' 32.4'
276'
>
15 i
LEVELS OF PLASMA LH in fertile women using 0.05 mg ethynylcstradiol or 0.06 -0.075 mg mestnnol and 0.5 mg norgertrol or 2.5 mg lynestrenol or 4 mg norethindrone acetate or 10 mg norethindrone for 5 to 12 yrs. by menstrual cycle day.
Eugynon & Ovral Anovlar
Fig. 4B. Levels
A
0
Ortho
Novum
Lindiol
of plasma LH in fertile women using 0.05 mg. of ethynylestradiol mg. of mestranol and 0.5 mg. of norgestrel or 2.5 mg. of lynestrenol or 4 mg. acetate or 10 mg. of norethindrone for 5 to 12 years, by menstrual cycle day.
at 0.05 > P > 0.02 except for the last cycle. With the 80 pg of EE and 100 pg of mestranol combinations a further decrease in the already low FSH level was not observed. Further suppression either was not feasible or its detection was beyond the sensitivity of the radioimmunoassay. Analysis of variance was applied, by dose and by cycle, to the data of cycles 7 to 12. For each of the five groups, the median FSH level was stable throughout the six successive cycles. No difference in the median FSH values (i.e., the degree of suppression) could be detected in comparing the three mestranol dose levels with each other, nor in comparing the two EE dose levels with each other. Finally, comparison of mestranol with EE combinations at the 50 or 80 pg estrogen level also showed no significant differences. It therefore appears that all these estrogen-progestin combinations show the maximum degree of FSH suppression at the lowest estrogen level tested. These results are consistent
10 mg 0
0
or 0.06 to 0.075 of norethindrone
with data based on ovulation inhibitionI where far lower estrogen dosage in combination with various progestins proved to be as effective as higher doses. LH levels. A. CONTROL DATA. The median LH values of the IUD group showed an unexplained variation during the first six cycles. These data were therefore excluded, and the LH va.lues of the pretreatment cycles of each group assigned to a particular estrogen regimen served as its own control. However, this greatly reduced the sample size of the controls and limited the comparisons which could be made with the treatment values. In any event, they were consistent with the two-way analysis of variance, which has a greater analytical power under these circumstances. B. ESTROGEN ADMINISTRATION (FIG. 2). Compared to its own pretreatment level, each of the mestranol and EE regimens showed a higher median LH value in cycle 1. From then on, there was a highly signifi-
Table Il. Frequency distribution contraceptives for 5 to 12 years
of FSH and LH values in women using high- or low-estrogen (samples taken randomly throughout the cycle)
samples at various FSH levels < 1.2 rnl.U./ml. / > 1.2 < 10.0 mI.U./ml. / > 10 mI.lJ./ml. 30 100 25 28 114 10 Number of samples at various LH levels Number
Estrogen
level
~
Low-dose High-dose I Estrogen
level
Low-dose High-dose
Q
0.4
mI.lJ./ml.
of
/
>
0.4
5.0
mI.U./ml.
5 8
oral
Total A’o. samples
of
155 1.5” Total No. samples
of
125 132
subjects. Particularly interesting in Figs. 3A and 38 are the pulses of FSH and LH (not necessarily concurrent) which are unassociated with the expected time of ovulation. There is an intimation of this phenomenon in some published longitudinal studies of normal cycles, but without any estimate of its frequency. From a practical point of view, such information is important, since diagnostic studies including a single high plasma gonadotropin value (at a nonovulatory time) might be misinterpreted if the normal occurrence of isolated gonadotropin pulses is not recognized. Plasma gonadotropin levels in long-term oral contraceptive users have not been investigated to any extent, although post-treatment return of ovulatory activity (implying normal gonadotropin dynamics) has been extensively documented.lg The plasma FSH and LH levels obtained at random times during treatment cycles of women who had been steroidal contraceptive users for 5 to 12 years are displayed in Figs. 4A and +B. Due to the relatively small number of samples, the data were grouped into low, intermediate, and high FSH and LH levels, and analyzed by the chi-square method. The data are subdivided into those from low-estrogen (50 pg of EE or 60 to 75 pg of mestranol) and higher estrogen (100 pg of mestranol) users (Table II). The frequency distribution of FSH values between low- and high-estrogen users differs only with respect to values of 10 m1.U. or above. More of these values were seen in the low-estrogen group. This difference is significant at P < 0.05. The higher values in the low-estrogen users are randomly distributed throughout the treatment cycle and occur with all the various formulations. The small number of samples does not permit analysis of the influence of different progestins. No significant difference was seen in the distribution of the two sets of LH values.
Volumr
122
Sumbrr
5
Comment These studies with oral EE and mestranol of controlled bioavailability” reveal novel effects on gonadotropin dynamics during 21 day cycles of administration, recognizing that only changes in the last week of each treatment cycle are being measured. The first cycle of estrogen exposure produced no significant change of the median FSH value at any dose of either estrogen. In the second cycle, a dose-related suppression appeared, and remained stable at each dose level for the succeeding four cycles. However, no significant suppression occurred at any time with 50 ,ug of mestranol, and the response to 50 pg of EE was equivocal. Mestranol and EE appeared to be equipotent over the 50 to 80 pg dosage range. The addition of any one of three synthetic progestins to the cyclic estrogen dosage produced a further prompt and consistent suppression of plasma FSH. No qualitative difference could be detected between the progestins at this profound level of suppression nor was there any difference between the various estrogen dosages. The effects on median LH values were quite different, for they rose by the latter part of the first treatment cycle at all dose levels, and then over the next five cycles they underwent a continuous decline which was related to the magnitude of the estrogen dose. EE appeared to be slightly more potent than mestranol at the 50 pg per day level, but at 80 pg per day no difference could be detected. The addition of progestins to the cyclic estrogen treatment produced a substantial further depression of median LH values, just as was seen with FSH, but in this case the level of mestranol seemed to make some difference in the degree of LH suppression. These findings differ from previous observations of the effect of ethynyl estrogens in castrate, postmenopausal, or normal cycling women. The dosage regimens and the time of measurement are quite different, and it is not possible to explain or reconcile these differences in the present state of our knowledge of estrogen-related gonadotropin dynamics; however, the findings give some insight into the relative
Ethynyl
estrogens
in oral
contraceptives.
III
635
potency of the two ethynyl estrogens and the magnitude of their effect in the range commonly used in contraceptive agents. The synergistic effect of the progestins, implicit in the high antiovulatory effectiveness of very low-dose estrogen-progestin combinations, is confirmed by these studies of concomitant gonadotropin levels. The randomly timed samples taken in long-term users of combination oral contraceptives show comparable or perhaps slightly greater degrees of gonadotropin suppression, with a suggestion of less FSH inhibition with the lower estrogen dose agents. What is more interesting, however, is the occurrence of relatively frequent pulses of gonadotropin release. This finding provides further evidence, if any is needed, that the pituitary is not suppressed by these agents in the same sense that the thyroid or the adrenal cortex are suppressed by chronically administered thyroxine or cortisone with resultant cellular involution and even atrophy. Cyclic estrogen-progestin therapy seems, rather, barely to keep gonadotropin release in check, preventing primarily the ovulatory surge (in a very high percentage of cycles) and reducing the general level of gonadotropin most of the time, while permitting occasional surges of gonadotropin output. This phenomenon, currently unexplained, is also seen in the normal cycles of IUD users, and appears to occur with some frequency. In the context of our related studies on the relative potency of ethynyl estradiol and mestranol,llq I5 these results confirm the approximate equipotency of these two compounds, in contrast to most investigations in other nonprimate species.
We wish to acknowledge the participation of Dr. Louis E. Moses, who was clinical supervisor of this project prior to his untimely death, the collaboration of Drs. Edris Rice-Wray and Jaime Gorodovsky of the Asociacion Pro-Salud Maternal, Mexico City, the extensive statistical assistance of Mr. Tazewell Dozier, the technical assistance of Mss. Joyce Mandel and Donna Shannon, and a generous gift of mestranol from Syntex Laboratories.
REFERENCES
1. Hagino, N., and Goldzieher, J. W.: Contraception 1: 131, 1970. 2. Wise, A. J., Gross, M. A., and Schalch, D. S.: J. Lab. Clin. Med. 81: 28, 1973. 3. Franchimont, P., Legros, J. J., and Meurice, J.: Harm. Metab. Res. 4: 288, 1972.
4. Yen, S. S., and Tsai, C. C.: J. Clin. Endocrinol. 33: 882, 1971. 5. Wallach, E. E., Root, A. W., and Garcia, C-R.: J. Clin. Endocrinol. 31: 376, 1970. 6. Crystle, C. D., Sawaya, G. A., and Stevens, V. C.: AM. J. OBSTET. GYNECOL. 116: 616, 1973.
7. 8. 9. 10. Il. 12.
13.
Stevens, V. C., and Vorys, N.: Obstet. Gynecol. Sum. 22: 781, 1967. Stevens, V. C.. Goldzieher, J. W., and Vorys, N.: AM. J. OBSTET. GYNECOL. 102: 95, 1968. Swerdloff, R. S., and Odell, W. D.: J. Clin. Endocrinol. 29: 157, 1969. Cargille, C. M., Vaitukaitis, J. L., Bermudez, J. A., and Ross, G. T.: J. Clin. Endocrinol. 36: 87, 1973. Tsai, C. C., and Yen, S. S.: J. Clin. Endocrinol. 33: 917, 1971. Vaitukaitis, J. L., Bermudez, J. A., Cargille, C. M., Lipsett, M. B., and Ross, G. T.: J. Clin. Endocrinol. 32: 503, 1971. Goldzieher, J. W., Kleber, J. W., Moses, L. E., and Rathmacher, R. P.: Contraception 2: 225, 1970.
14.
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16. 17. 18. 19.
Goldzieher, J. W., Maqueo, M., Chenault, C. B., and Woutersz, T. B.: AM. J. OBSTET. GYNECOL. 122: 615, 1975. Goldzieher, J. W., de la Pefia, A., Chenault. C. I~.> and Woutersz. T. B.: AM. J. OBSTET. GYNECOL. 122: 619, 1975. Odell, W. D., Ross, G. T., and Rayford, P. L.: J. Lab. Clin. Med. 70: 973, 1967. Midgley, A. R.: J. Clin. Endocrinol. 27: 295, 1967. Rodbard, D., Rayford, P. L., Cooper, J. A., and Ross, G. T.: J. Clin. Endocrinol. Metab. 28: 1412. 1968. Rice-Wray, E., Correu, S., Gorodovsky, J., Esquivel, J., and Goldzieher, J. W.: Fertil. Steril. 18: 212, 1967.
