CONTRACEPTION
SOME ESTROGENIC EFFECTS OF TWO ORAL CONTRACEPTIVES CONSISTING NORGESTREL AND TWO DIFFERENT DOSES OF ETHYNYLESTRADIOL
J.F. Miller, D.M., M.R.C.O.G. * Consultant Obstetrician and Gynaecologist, Hospital, Southampton SO9 4XY
K.Fotherby, PhD., F.R.I.C. Department of Steroid Biochemistry, School, London
P.G.T. Senior
OF
Southampton
General
Royal Postgraduate
Medical
Bye, M.B., D. Pharm. Med. Medical Adviser, Schering Chemicals
Max Elstein, M.D., M.R.C.O.G. Professor of Obstetrics and Gynaecology, South Manchester
University
Hospital
of
ABSTRACT Some biological and biochemical effects (ie. KPI, cervical mucus, SHBG and ceruloplasmin) as well as serum ethynylestradiol and serum following the use of two oral contraceptives containing norgestrel, the same amount of norgestrel (dl-norgestrel 0.5 mg) and either 30 Pg or 50 pg of ethynylestradiol were compared. There was no difference in the clinical features in either group of patients. There was a statistically significant difference in the levels of unconjugated ethynylestradiol but not in the other biochemical parameters studied. It is concluded that the absence of any such difference might be attributable to the strong antiestrogenic effects of dl-norgestrel
Accepted
Requests
for
publication
for reprints
June
4,
1979
from:
J.F. Miller, D.M., M.R.C.O.G. * Consultant Obstetrician and Gynaecologist, Hospital, Southampton SO9 4XY
JULY
1979 VOL. 20 NO. 1
Southampton
General
5
CONTRACEPTION
INTRODUCTION Since the British Committee on Safety of Medicines recommended in 1969 (l)that the dose of estrogen used in oral contraceptives should not exceed 5Opg, the estrogen content in some preparations has been reduced. Epidemiological evidence of any increased safety of these newer formulations with less estrogen is still awaited. They have been shown however, to be acceptable to most women (2). In addition, pituitary function returns rapidly after discontinuing the preparations (3). Although many effects of estrogens in the human are known, few studies of dose-effect relationships with estrogens used either alone or in combination with gestagens in oral contraceptives have been performed. The reported ability of the progestogen, norgestrel, to antagonise the effect of the estrogen component on certain parameters (4) requires that the estrogenic effects of such combinations should be directly measured, and not inferred from dose-effect studies using estrogens alone. Ceruloplasmin and sex hormone binding globulin (SHBG) are plasma proteins that are regarded as suitable parameters of estrogenicity. A dose-effect study of SHBG with two oral contraceptives containing the same hormones and differing only in their estrogen content does not seem to have been carried out. The purpose of this study was to determine whether a reduction in ethynylestradiol dosage from 5Opg to 30&g was associated with any change in the biological or biochemical parameters.
MATERIAL AND METHODS Eleven healthy women, age 18-25 years, with normal menstrual cycles were subjects took the contraceptives studied for six months. Alternate containing 3Opg or 50&g ethynylestradiol (Eugynon 30 and Eugynon 50) (Schering Chemicals Ltd)). The tablets were taken regularly in the morning each day during the course of tablets and the patients were seen for examination and venepuncture on the last day of the first and sixth course of tablets. However, where this was not possible, in two subjects who took their tablets in the evening, they had their venepunctures the
6
JULY
1979 VOL. 20 NO. 1
CONTRACEPTION
following morning to ensure a similar ingestion-sampling interval. Six volunteers were given Eugynon 30 and the remaining five Eugynon 50. There were no significant differences in the mean age, height, weight or parity of the two groups. The biological features of estrogenic effects in the genital tract were assessed at each visit by examining the karyopyknotic index (KPI) of the vaginal wall smear and by observing the ferning, spinnbarkeit, volume and clarity of the cervical mucus (5). Venepuncture occurred 12 - 15 hours after tablet administration and 10 ml of venous blood was taken. After clotting, the serum was separated, frozen and stored at -2O'C until analysed. Assays were performed for unconjugated and conjugated ethynylestradiol (6),dlnorgestrel (7), SHBG activity (8) and ceruloplasmin (9). The crossreaction of norgestrel in the EE assay was 0.03%. The recoveries from the methods were EE 602 10% (mean 2 SD) and for norgestrel 90 ?; 6%. The intra- and inter-assay coefficients of variation for both assays were less than 15% and the sensitivity of both assays was about 50 pg/ml.
RESULTS The KPI ranged between 1.5% and 22%. There was no correlation of the KPI with time or with the amount of ethynylestradiol contained in the pill. Cervical mucus could not be obtained on seven occasions, approximately one third of examinations, because the volume was so scanty. When a sample of mucus was obtained, the volume was always low, twice being 0.2 ml and on the remaining 13 occasions less than 0.1 ml. On no occasion was the spinnbarkeit greater than 1 cm and on seven occasions the mucus was so "tacky" that no spinnbarkeit could be demonstrated. Ferning was not seen in any specimen. There were no differences between the characteristics of the cervical mucus from women receiving the two formulations or between the samples taken at the end of the first and sixth courses. The biochemical results are shown in the Table. The mean ethynylestraand norgestrel concentrations were higher in patients taking diol Eugynon 50. At six months the mean conjugated ethynylestradiol and norgestrel levels tended to be lower than at one month, but the differences were not significant. The mean SHBG activity and ceruloplasmin levels tended to be higher at six months. The only values between which there were any statistically significant difference were those of unconjugated ethynylestradiol when the two different dosage regimes were compared (~(0.01). There were no statistically significant differences between the other biochemical parameters either in respect of the duration of administration or in respect of the two different dosage regimes.
JULY
1979 VOL. 20 NO. 1
7
CONTRACEPTION
Table. Results of Biochemical Estimations
lean value C&D) at times after starting treatment Ethynylestradiol dose ().%/day)
8
1 month
6 months
Unconjugated 1) ethynylestradiol (pg/ml
30 50
63 109
(18) (32)
60 103
(35) (31)
Conjugated 1) ethynylestradiol (pg/ml
30 50
277 419
(145) (149)
170 271
(45) (178)
Total 1) ethynylestradiol (pg/ml
30 50
400 528
(156) (168)
230 406
(69) (190)
dl-norgestrel (pg/ml)
30 50
2052 (627) a299 (608)
1611 2200
(451) (527)
Sex hormone binding globulin activity (n mol/ml)
30 50
).47 (0.08) I.45 (0.10)
0.52 (0.06) 0.49 (0.05)
Ceruloplasmin (mu/ml)
30 50
582 685
(119) (130)
JULY
672 717
(70) (178)
1979 VOL. 20 NO. 1
CONTRACEPTION
DISCUSSION The only statistically significant difference in the biological features between the two groups of patients in this study was the expected difference in the plasma concentrations of unconjugated ethynylestradiol. However the absence of any difference between the two formulations might be explained by the strong anti estrogenic effect of norgestrel(4). The lack of a significant difference at one and six months might suggest that a steady state had been reached by the end of the first month. This early equilibration has been reported previously by Musa -et al.(lO).These similarities suggest that it is unlikely that there would be any major differences in biological effect between the combination containing 3Opg of ethynylestradiol when compared to the 50&g preparation. This has been borne out in clinical practice. Epidemiological evidence of any possible change in the rate of thromboembolic disease with this lower estrogen dosage is eagerly awaited. It is concluded that there are no significant changes in the parameters measured but that the strong anti:estrogenic effect of norgestrel may be masking any effect of the different amounts of ethynylestradiol.
JULY
1979 VOL. 20 NO. 1
9
CONTRACEPTION REFERENCES 1.
Scowen E.F. Oral contraception containing.oestrogens. British Medical Journal 5, 744 (1969)
2.
Bye P.G.T. and Elstein M. Clinical assessment of a low oestrogen combined oral contraceptive. British Medical Journal I, 389 (1973)
3.
Elstein, M., Morris S.E., Groom G.V., Jenner D.A., Scarisbrick J.J. and Cameron E.H.D. Studies on low-dose oral contraceptives: cervical mucus and plasma hormone changes in relation to circulating d-norgestrel and 17a ethynyl estradiol concentrations. Fertility and Sterility 27, 892 (1976)
4.
Briggs M. Effects of oral progestogens on estrogen-induced changes in serum protein. In the Second International Norgestrel Symposium, Excerpta Medica, Amsterdam 1974 p. 35
5.
Elstein M. The cervix and its mucus in the management of infertility. In Clinics in Obstetrics and Gynaecology, Edited by I.D. Cooke, W.B, Saunders 6 Co. Ltd., London, 1974, p. 345
6.
Warren R.J. and Potherby K. Radioimmunoassay of ethynylestradiol. Journal of Endocrinology 63, 3Op (1974)
7.
Warren R.J. and Fotherby K. Radioimmunoassay of synthetic progestogens, norethisterone and norgestrel. Journal of Endocrinology 62, 605 (1974)
8.
Rosner W. A simplified method for the quantitative determination of testosterone-es.tradiol-binding globulin activity in human plasma, Journal of Clinical Endocrinology and Metabolism 3, 983 (1972)
9.
Schosinsky K.H., Lehmann H.P. and Beeler M.F. Measurement of ceruloplasmin from its oxide activity in serum by use of Q-dianisidine dihydrochlori'de, Clinical Chemi‘stry0, 1556 (1974)
10.
10
Musa B.U., Seal V.S. and Doe R.P. Evaluation of certain plasma proteins in man following estrogen administration: A dose-response relationship. Journal of Clinical Endocrinology _25, 1163 (1965)
JULY
1979 VOL. 20 NO. 1
SOME ESTROGENIC EFFECTS OF TWO ORAL CONTRACEPTIVES CONSISTING NORGESTREL AND TWO DIFFERENT DOSES OF ETHYNYLESTRADIOL
J.F. Miller, D.M., M.R.C.O.G. * Consultant Obstetrician and Gynaecologist, Hospital, Southampton SO9 4XY
K.Fotherby, PhD., F.R.I.C. Department of Steroid Biochemistry, School, London
P.G.T. Senior
OF
Southampton
General
Royal Postgraduate
Medical
Bye, M.B., D. Pharm. Med. Medical Adviser, Schering Chemicals
Max Elstein, M.D., M.R.C.O.G. Professor of Obstetrics and Gynaecology, South Manchester
University
Hospital
of
ABSTRACT Some biological and biochemical effects (ie. KPI, cervical mucus, SHBG and ceruloplasmin) as well as serum ethynylestradiol and serum following the use of two oral contraceptives containing norgestrel, the same amount of norgestrel (dl-norgestrel 0.5 mg) and either 30 Pg or 50 pg of ethynylestradiol were compared. There was no difference in the clinical features in either group of patients. There was a statistically significant difference in the levels of unconjugated ethynylestradiol but not in the other biochemical parameters studied. It is concluded that the absence of any such difference might be attributable to the strong antiestrogenic effects of dl-norgestrel
Accepted
Requests
for
publication
for reprints
June
4,
1979
from:
J.F. Miller, D.M., M.R.C.O.G. * Consultant Obstetrician and Gynaecologist, Hospital, Southampton SO9 4XY
JULY
1979 VOL. 20 NO. 1
Southampton
General
5
CONTRACEPTION
INTRODUCTION Since the British Committee on Safety of Medicines recommended in 1969 (l)that the dose of estrogen used in oral contraceptives should not exceed 5Opg, the estrogen content in some preparations has been reduced. Epidemiological evidence of any increased safety of these newer formulations with less estrogen is still awaited. They have been shown however, to be acceptable to most women (2). In addition, pituitary function returns rapidly after discontinuing the preparations (3). Although many effects of estrogens in the human are known, few studies of dose-effect relationships with estrogens used either alone or in combination with gestagens in oral contraceptives have been performed. The reported ability of the progestogen, norgestrel, to antagonise the effect of the estrogen component on certain parameters (4) requires that the estrogenic effects of such combinations should be directly measured, and not inferred from dose-effect studies using estrogens alone. Ceruloplasmin and sex hormone binding globulin (SHBG) are plasma proteins that are regarded as suitable parameters of estrogenicity. A dose-effect study of SHBG with two oral contraceptives containing the same hormones and differing only in their estrogen content does not seem to have been carried out. The purpose of this study was to determine whether a reduction in ethynylestradiol dosage from 5Opg to 30&g was associated with any change in the biological or biochemical parameters.
MATERIAL AND METHODS Eleven healthy women, age 18-25 years, with normal menstrual cycles were subjects took the contraceptives studied for six months. Alternate containing 3Opg or 50&g ethynylestradiol (Eugynon 30 and Eugynon 50) (Schering Chemicals Ltd)). The tablets were taken regularly in the morning each day during the course of tablets and the patients were seen for examination and venepuncture on the last day of the first and sixth course of tablets. However, where this was not possible, in two subjects who took their tablets in the evening, they had their venepunctures the
6
JULY
1979 VOL. 20 NO. 1
CONTRACEPTION
following morning to ensure a similar ingestion-sampling interval. Six volunteers were given Eugynon 30 and the remaining five Eugynon 50. There were no significant differences in the mean age, height, weight or parity of the two groups. The biological features of estrogenic effects in the genital tract were assessed at each visit by examining the karyopyknotic index (KPI) of the vaginal wall smear and by observing the ferning, spinnbarkeit, volume and clarity of the cervical mucus (5). Venepuncture occurred 12 - 15 hours after tablet administration and 10 ml of venous blood was taken. After clotting, the serum was separated, frozen and stored at -2O'C until analysed. Assays were performed for unconjugated and conjugated ethynylestradiol (6),dlnorgestrel (7), SHBG activity (8) and ceruloplasmin (9). The crossreaction of norgestrel in the EE assay was 0.03%. The recoveries from the methods were EE 602 10% (mean 2 SD) and for norgestrel 90 ?; 6%. The intra- and inter-assay coefficients of variation for both assays were less than 15% and the sensitivity of both assays was about 50 pg/ml.
RESULTS The KPI ranged between 1.5% and 22%. There was no correlation of the KPI with time or with the amount of ethynylestradiol contained in the pill. Cervical mucus could not be obtained on seven occasions, approximately one third of examinations, because the volume was so scanty. When a sample of mucus was obtained, the volume was always low, twice being 0.2 ml and on the remaining 13 occasions less than 0.1 ml. On no occasion was the spinnbarkeit greater than 1 cm and on seven occasions the mucus was so "tacky" that no spinnbarkeit could be demonstrated. Ferning was not seen in any specimen. There were no differences between the characteristics of the cervical mucus from women receiving the two formulations or between the samples taken at the end of the first and sixth courses. The biochemical results are shown in the Table. The mean ethynylestraand norgestrel concentrations were higher in patients taking diol Eugynon 50. At six months the mean conjugated ethynylestradiol and norgestrel levels tended to be lower than at one month, but the differences were not significant. The mean SHBG activity and ceruloplasmin levels tended to be higher at six months. The only values between which there were any statistically significant difference were those of unconjugated ethynylestradiol when the two different dosage regimes were compared (~(0.01). There were no statistically significant differences between the other biochemical parameters either in respect of the duration of administration or in respect of the two different dosage regimes.
JULY
1979 VOL. 20 NO. 1
7
CONTRACEPTION
Table. Results of Biochemical Estimations
lean value C&D) at times after starting treatment Ethynylestradiol dose ().%/day)
8
1 month
6 months
Unconjugated 1) ethynylestradiol (pg/ml
30 50
63 109
(18) (32)
60 103
(35) (31)
Conjugated 1) ethynylestradiol (pg/ml
30 50
277 419
(145) (149)
170 271
(45) (178)
Total 1) ethynylestradiol (pg/ml
30 50
400 528
(156) (168)
230 406
(69) (190)
dl-norgestrel (pg/ml)
30 50
2052 (627) a299 (608)
1611 2200
(451) (527)
Sex hormone binding globulin activity (n mol/ml)
30 50
).47 (0.08) I.45 (0.10)
0.52 (0.06) 0.49 (0.05)
Ceruloplasmin (mu/ml)
30 50
582 685
(119) (130)
JULY
672 717
(70) (178)
1979 VOL. 20 NO. 1
CONTRACEPTION
DISCUSSION The only statistically significant difference in the biological features between the two groups of patients in this study was the expected difference in the plasma concentrations of unconjugated ethynylestradiol. However the absence of any difference between the two formulations might be explained by the strong anti estrogenic effect of norgestrel(4). The lack of a significant difference at one and six months might suggest that a steady state had been reached by the end of the first month. This early equilibration has been reported previously by Musa -et al.(lO).These similarities suggest that it is unlikely that there would be any major differences in biological effect between the combination containing 3Opg of ethynylestradiol when compared to the 50&g preparation. This has been borne out in clinical practice. Epidemiological evidence of any possible change in the rate of thromboembolic disease with this lower estrogen dosage is eagerly awaited. It is concluded that there are no significant changes in the parameters measured but that the strong anti:estrogenic effect of norgestrel may be masking any effect of the different amounts of ethynylestradiol.
JULY
1979 VOL. 20 NO. 1
9
CONTRACEPTION REFERENCES 1.
Scowen E.F. Oral contraception containing.oestrogens. British Medical Journal 5, 744 (1969)
2.
Bye P.G.T. and Elstein M. Clinical assessment of a low oestrogen combined oral contraceptive. British Medical Journal I, 389 (1973)
3.
Elstein, M., Morris S.E., Groom G.V., Jenner D.A., Scarisbrick J.J. and Cameron E.H.D. Studies on low-dose oral contraceptives: cervical mucus and plasma hormone changes in relation to circulating d-norgestrel and 17a ethynyl estradiol concentrations. Fertility and Sterility 27, 892 (1976)
4.
Briggs M. Effects of oral progestogens on estrogen-induced changes in serum protein. In the Second International Norgestrel Symposium, Excerpta Medica, Amsterdam 1974 p. 35
5.
Elstein M. The cervix and its mucus in the management of infertility. In Clinics in Obstetrics and Gynaecology, Edited by I.D. Cooke, W.B, Saunders 6 Co. Ltd., London, 1974, p. 345
6.
Warren R.J. and Potherby K. Radioimmunoassay of ethynylestradiol. Journal of Endocrinology 63, 3Op (1974)
7.
Warren R.J. and Fotherby K. Radioimmunoassay of synthetic progestogens, norethisterone and norgestrel. Journal of Endocrinology 62, 605 (1974)
8.
Rosner W. A simplified method for the quantitative determination of testosterone-es.tradiol-binding globulin activity in human plasma, Journal of Clinical Endocrinology and Metabolism 3, 983 (1972)
9.
Schosinsky K.H., Lehmann H.P. and Beeler M.F. Measurement of ceruloplasmin from its oxide activity in serum by use of Q-dianisidine dihydrochlori'de, Clinical Chemi‘stry0, 1556 (1974)
10.
10
Musa B.U., Seal V.S. and Doe R.P. Evaluation of certain plasma proteins in man following estrogen administration: A dose-response relationship. Journal of Clinical Endocrinology _25, 1163 (1965)
JULY
1979 VOL. 20 NO. 1
