Effect of a combination of norethynodrel and mestranol on plasma luteinizing hormone in normal women

Effect of a combination

of norethynodrel

mestranol on plasma luteinizing

and

hormone in

normal women G.

E.

ABRAHAM,

E.

L.

KLAIBER,

D.

BROVERMAN,

Shrewsbury,

M.D. M.D. PH.D.

Massachusetts

Plasma luteiniring hormone (LH) leuels were studied by radioimmunoassay two consecutive menstrual cycles in 6 normal subjects. The first cycle served control cycle. The subjects received a combination of norethynodrel (5 mg.) mestranol (0.075 mg.) daily during the following cycle. A midcycle surge of LH was observed in every subject during the control cycle, but in none during treated cycle. The basal levels of LH during the treated cycles were significantly lower than during the follicular phase of the control cycles. The mechanisms which this oral contraceptive drug affects plasma LH are discussed.

S I N c E Yalow and Bersonl reported their sensitive radioimmunoassay for insulin in human plasma, this method has been applied to almost every polypeptide hormone.“? 3 The impact of this powerful tool is just beginning to be felt in the field of obstetrics and gynecology. Old concepts are being re-evaluated and new fields of investigation have become possible. Until recently, the secretion of human luteinizing hormone (LH) could be measured only indirectly by bioassay4 in urinary extractP and a midcycle peak has been observed. Because of the insensitivity of this bioassay, a large pool of urine had to be extracted. As a consequence, the exact time of this LH peak could not be ascertained. Bioassay techniques have also been applied to the measurement of LH in plasma,g-12 but because a large volume of plasma pooled from several individuals was required, the study of plasma LH during the menstrual From the Worcester Foundation Experimental Biology. This study was supported Health Service Training NIMH 5TI MHI0625.

for

by Public Grant No.

during as u and

plasma the by

cycle became impractical. Recently, the radioimmunoassay technique which can be performed directly without extraction on 0.05 to 0.2 ml. of plasma or serum has been applied to the measurement of plasma JJp-17 and the pattern of LH in plasma during the normal menstrual cycle has been established. The midcycle peak of LH in urine reported previously5-8 was also found in plasma. Ross, Odell, and Rayfordls and Schalch found significantly lower and associates’” levels of plasma LH in the luteal than in tht follicular phase of the menstrual cycle, hut this finding could not be confirmed by others.lSy I6 The midcycle surge of plasma LH found in every normal menstrual cycle studied by radioimmunoassay was suppressed by an oral contraceptive drug (a combination of norethynodrel, 2.5 mg., and mestranol, 0.1 mg. J in every one of the subjects studied by ROSS, Odell, and Rayford. I3 This confirmed reports of a suppression of the midcycle increase in urine of LH activity as measured by bioassay. 5l ?O The same author99 also reported a 1038

Volume Number

104 7

Table I. Clinical

Norethynodrel

and mestranol

1039

data Cycle length control

Cycle length Enovid

Prepeak duration

Day of

Postpeak

Subjects

Parity

H. M.

o-o-o-o

31.

25

17

18

duration 13

g M. G. E. G. N. G.

o-o-o-o o-o-o-o o-o-o-o o-o-o-o

25 30 32 30 31

2.5 25 24 25 28

11 16 17 15 17

12 17 18 16 18

13 14 14 13

lower basal LH level during the treated cycle when compared with the mean follicular level in the same subject. This latter finding is novel and was never suspected by investigators using bioassay. This effect of an oral contraceptive drug on the basal level of LH in plasma could be due to either a decrease in secretion rate by the pituitary gland or to an increased metabolic clearance rate (MCR) which would result in a lower plasma level without any change in secretion rate. Kohler, Ross, and Ode11,21 using Tait and Burstein’s2? method for measuring the metabolic clearance rate and production rate compared two groups of subjects: one control group composed of normal premenopausal women and the other group consisting of normal premenopausal subjects on an oral contraceptive drug (norethynodrel and mestranol at a dose not reported). They found no difference in MCR of plasma LH when the two groups were compared, whereas the subjects on the oral contraceptive drug showed much lower plasma LH levels. They measured the production rate of LH and found it to be significantly lower in the treated group. Those values, expressed in milliunits of 2nd International Reference Preparation of human menopausal gonadotropin per minute were: 734 + 170 S.E. (5 control subjects) and 387 f 86 S.E. (5 treated subjects). Although the above study suggests a possible site of action of the oral contraceptive drugs, the blockage of ovulation by a decreased production rate of pituitary LH, an extensive review by Diczfalusy23 on the mode of action of contraceptive drugs showed some

I LH

peak

interesting findings. One, among others, was that the bIockage of ovulation is not a necessary condition for the contraceptive effects of such drugs; different drugs and the same drug at different dose levels may exert their contraceptive effects by different modes and sites of action. The study here reported repeats the study done by Ross, Odell, and Rayfordls with the same contraceptive drug at a different dose level. Material

and

methods

Six normally menstruating, young, adult women were followed during two consecutive cycles. The clinical data on these subjects are shown in Table I. The first cycle served as a control cycle, and a combination of norethynodrel (5 mg.) and mestranol (0.075 mg.), kindly supplied by G. D. Searle & Company, was administered on day 5 of the next cycle and continued for 20 days. Daily basal body temperatures and measurement of the urinary excretion of pregnanediol on days 10 and 20 of each cycle served as indirect indications of ovulation. Using the above criteria, all subjects showed signs of ovulation during the control cycles and absence of ovulation during the medicated cycles. Blood was drawn daily by venipuncture into an oxalated container and the plasma separated by centrifugation. Plasma samples were kept at -20’ C. until the LH was determined by radioimmunoassay as described by Odell, Ross, and Rayford.13 The specificity of the antiserum* used has been demonstrated.24 In our laboratory the sensitivity of “The authors help.

antiserum wish also

was supplied by Dr. W. D. Odell. The to thank Dr. Ode11 for his invaluable

1040

Abraham,

Klaiber,

and

Broverman

NGM

HLH

25

.I

5

I

2.5

5

I

.5

I

2

5 Mill1

IO Units

20 HCG

50

100

+ IRPHMG

IO I

200 2@

Fig. 1. Dose-response curve with three different preparations as standards. 76 PPD denotes the per cent of I?“I-LH precipitated (bound) with 100 per cent used as the proportion of IT-LH bound when no standard preparation was added. A mass of 0.3 ng. of l?zI-LH was used.

the method was 0.1 nanogram (ng.) of HLH* when 0.05 - 0.1 ng. of Y-HLH was used. Since 0.2 ml. of plasma was used for the assay, the sensitivity was therefore 0.5 ng. per milliliter of plasma. The within-assay variance was studied by duplicate runs and varied from 3 to 8.6 per cent (coefficient of variation). On two occasions, the betweenassay variance was greater: 14.8 and 16.5 per cent, respectively. For this reason, all samples from the same subjects were run in the same batch. Since the viscosity of the incubation media affects the time required to achieve equilibrium, 0.2 ml. of cow plasma was routinely added to the test tubes used for the standard curve. Cow LH does not interfere with this assay.

the pituitary (HLH) and the urinary (2nd IRP-HMG) * preparations were compared. Postmenopausal plasma gave an identical slope with the pituitary preparation. For this reason, the pituitary HLH was used for radioiodination and as a standard. At the 50 per cent displacement of labeled Y-HLH, 1 ng. of HLH was equivalent to 5 mu. of 2nd IRP-HMG. An LH peak was obtained in every subject during the control cycle, but in none during the Enovid-treated cycle. Plasma LH values in normal and Enovid-treated cycles yielded a distribution as shown in Fig. 2. When

the

LH

values

from

the

follicular-

Fig. 1 shows a dose-response curve with three different preparations of human origin. The steepness of the slope was different when

phase were compared with those of the luteal phase (Table II) : the follicular values were significantly higher in one subject. However, in all of the subjects the follicular values were significantly higher than the Enovid cycle values. The plasma LH levels WC’W higher in the luteal phase than in the Enovid

“HLH Pituitary

‘Obtained Institute of

Results

= HLH-LER-822-2, Agency, Baltimore,

obtained Maryland.

from

the

National

from Medical

the Medical Research,

Research Mill Hill,

Council, London,

National England.

Volume Number

104 7

Norethynodrel

and mestranol

1041

;24 Control

:: aI6

-I2 Fig. 2. Scattergram 2nd IRP-HMG.

Table II. Plasma cycle and during Subjects

L. T. G. F. M. G.

E. G. N. G.

levels of LH during the treated cycle

f

standard

0

the follicular

Luteal*

4

Treated

cycle*

5.6 5 0.5 (10)

5.4 + 0.2

10.1 f 0.5 (11)

10.8 k 1.1 ( 9)

7.9 + 0.5

10.3 + 0.7 ( 9)

a.8 2 1.1 (11)

6.4 2 0.3

11.6 + 0.5 (13)

10.8 it 1.5 (10)

a.8 + 0.7

17.5 + 0.4 (12)

16.8 ? 1.1

16.3 + 0.3

(12)

(21)

16.9 t 0.6

14.0 fc 0.3 ( 8)

14.9 + 0.3 (19)

error;

( )

12 +

8

and luteal

7.8 lr 1.6 (10)

(12) ‘Mean

4

to L H peak Days in relation of plasma LH levels during the control and treated cycles. 1 ng. = 5 mu.

Follicular*

H. M.

8

cycles

=

No.

of

determinations;

phases of the control

Follicular vs. luteal

Follicular treated

Luteal vs. treated

vs.

N.S.

P < 0.05

N.S.

N.S.

P < 0.01

P < 0.02

N.S.

P < 0.001

P < 0.02

N.S.

P < 0.01

N.S.

N.S.

P < 0.02

N.S.

P < 0.01

P < 0.01

N.S.

(20) (1’3) (21) (20)

values

are

expressed

in

mU.

2nd

IRP-HMG

per

milliliter

of

plasma.

cycle in 2 subjects. Plasma LH values, excluding the peak levels, were significantly higher (P < 0.05) in young adult male than in female subjects (Fig. 3). Comment The most consistent findings in this study were the midcycle surge of plasma LH was completely suppressed by norethynodrel plus mestranol and the basal LH level during the treated cycle was lower than during the follicular phase.

The changes in plasma LH caused by the administration of 5 mg. of norethynodrel and 0.075 mg. of mestranol were the same as those produced by the administration of a combination of those same drugs at a different ratio and a different dose level.ls Although this does not rule out other sites of action, the decreased production rate of pituitary LH and the suppression of the midcycle surge of this hormone could be in themselves sufficient to confer a contraceptive effect on these drugs. What remains to be answered is

1042

Abraham,

Klaiber,

Moles

and

August I, 1969 Am. J. Obst. & Gym.

Broverman

Females L------A

(611

J

Fol

Lut

Enovid

(671

(601

(1161

whether or not conception occurring in subjects taking this oral contraceptive is the result of a failure of the drug in those subjects to suppress the midcycle surge of LH. Although it seems unlikely that ovulation could occur in some subjects in spite of the suppression of the LH surge, this possibility has not been completely ruled out. Whether the action of the norethynodrel-mestranol combination is on the synthesis or the release of LH is at present conjectural.

0

Fig. 3. Plasma

LH levels in male and female subjects. Numbers in parentheses = number of male subjects; number of determinations in 6 female subjects. Mean plasma LH values (bar height) + standard error.

REFERENCES

13

1. Yalow, R. S., and Berson, S. A.: J. Clin. Invest. 39: 119, 1960. 2. Felber, J. P.: Helvet. Med. Acta 33: 367, 1966. 3. Potts, J. T., Jr., Sherwood, L. M., O’Riordan, J. L. H., and Aurbach, G. D.: In Dock, W., and Snapper, I., editors: Advances in Internal Medicine, Chicago, 1967, Year Book Medical Publishers, vol. XIII, p. 183. 4. Parlow, A. F.: Fed. Proc. 17: 402, 1961. 5. McArthur, J., Worcester, J., and Ingersoll, F. M.: J. Clin. Endocrinol. 18: 1186, 1958. 6. Fukushima, M., Stevens, V. C., Gantt, C. L., and Vorys, N.: J. Clin. Endocrinol. 24: 205, 1964. 7. Becker, K. L., and Albert, A.: J. Clin. Endocrinol. 25: 962, 1965. 8. Rosenberg, E., and Keller, P. J.: J. Clin. 9. IO.

11. 12.

Endocrinol. 25: 1262, 1965. Apostolakis, M.: J. Endocrinol.

14.

and Reichlin, 1968. 15. 16.

17. 18. 19.

S.: J. Clin.

P. C.,

Invest. 47: 665,

Faiman, C., and Ryan, R. J.: J. Clin. Endocrinol. 27: 1711, 1967. Catt, K. J., Niall, H. D., Tregear, G. W., and Burger, H. G.: J. Clin. Endocrinol. 28: 121, 1968. Midgley, A. R., Jr.: Endocrinology 79: 10, 1966. Ross, G. T., Odell, W. D., and Rayford, P. L.: Science 155: 1679, 1967. Ross, G. T., Odell, W. D., and Rayford. P.

L.: Lancet 2: 1255, 1966. 20.

21.

19: 377, 1960.

McArthur, J., Antoniades, H., Larson, L., Pennell, R., Ingersoll, F., and Ulfelder, H.: J. Clin. Endocrinol. 24: 425, 1964. Louchart, J., Truffert, J., and DeCourt, J.: Acta endocrinol. 49: 293, 1965. Keller, P. J.: Acta endocrinol. 52: 341, 1966.

Odell, W. D., Ross, G. T., and Rayford, L.: J. Clin. Invest. 46: 248, 1967. Schalch, D. S., Parlow, A. F., Boon, R.

‘VI iL. 23. 2‘4.

Fukushima, M., Stevens, V. C., Gantt, C. L., and Vorys, N.: J. Clin. Endocrinol. 24: 205, 1964. Kohler, P. O., Ross, G. T., and Odell, W. D.: J. Clin. Invest. 47: 38, 1968. Tait, J. F., and Burstein, S.: Hormones 5: 441, 1964. Diczfalusy, E.: AM. J. OBST. & GYNEC. 100: 136, 1968. Ode& W. D., and Reichert, L. E.: Manuscript in preparation, 1968.