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Postovulatory contraception in women with large doses of norethindrone
POSTOVULATORY
CONTRACEPTION IN WOMEN WITH LARGE DOSES OF NORETHINDRONE
KarlCGsta
Nygren, Elof D.B. Johansson
The Department of Obstetrics and Gynecology University Hospital, Uppsala, Sweden
and Leif Wide
and the Department
of Clinical Chemistry,
ABSTRACT The postovulatory administration of large doses of norethindrone to women has previously been shown to drastically reduce the peripheral plasma levels of progesterone. In IO treatment cycles, compared to 10 control cycles from the same women, this effect was confirmed and a similar effect upon the peripheral plasma levels of estradiol was found. The contraceptive efficacy of this treatment was tested. Eighty volunteers were treated during 301 cycles. Eighty per cent of the women had been pregnant before. The plasma levels of progesterone and estradiol were determined before and after treatment in each cycle. A very sensitive test for the detection in serum of human chorionic gonadotrophin (HCG) was included. A total dose of 200 mg of norethindrone was used in two different schedules (100 mg daily on day 21 - 22 and 50 mg daily on day 19 - 22). Twenty-two pregnancies occurred, but in 4 of these women, the only indication of pregnancy was the detection of HCG. Two women did not take the tablets according to schedule, leaving 16 patients who had to be aborted therapeutically. At least 70 per cent of the cycles were found to be ovulatory. Control of vaginal bleeding was good and only minor side effects occurred. The treatment possibly reduced fertility in this group of women but the contraceptive efficacy was obviously too low to merit further use of the present dose and schedules.
Accepted
JUNE
1972
for
publication
A70L.
3; NO.
May 8,
6
1972.
445
CONTRACEPTION
INTRODUCTION Norethindrone (NET)* and other synthetic gestagens have been shown to drastically reduce the peripheral plasma levels of progesterone when given after ovulation to women (1). In preliminary reports (2, 3), postovulatory treatment with NET has also been claimed to depress the peripheral levels of estradiol. To verify this, an investigation was made concerning the effect of postovulatory treatment with NET upon the peripheral levels of both progesterone and estradiol. It was considered that the decreased periphera1 levels of the ovarian steroids might influence the development of the postovulatory endometrium and thereby impair the possibilities for normal implantation of the blastocyst. To investigate the contraceptive efficacy of such a treatment, NET was given postovulatory to pregnancy exposed women. As this treatment might result in a ” menstrual bleeding” that would, in fact, be an early abortion, a very sensitive test for the detection in serum of human chorionic gonadotrophin (HCG) (4) was included in the study. METHODS Study
A. Effect on the levels of progesterone
and estradiol
Subjects: Ten healthy women, 20 to 32 years of age, all employed volunteered for this study. All had regular menstrual cycles. Treatment: Various doses number of days (mean postovulatory day. The progesterone and estradiol
by the hospital,
of NET (mean 37 mg, range 5 100 mg) were given for various 4.3 days, range 2 6 days) starting on the third or fourth day of ovulation was calculated from the plasma levels of as described in previous papers ( 1, 5 ).
Samples: Daily blood samples for the assay of progesterone and estradiol veni puncture during the periovulatory and luteal phases of each cycle. Study
B. Contraceptive
were collected
by
efficacy
Subjects: Women seeking ccntraceptive advice at the University Hospital of Uppsala. Sweden, volunteered for this investigation. Those fulfilling the following criteria were accepted: I / Age 18 45 years. 2/ Good general health and normal findings upon gynecological examination, including a vaginal smear, palpation of the mammae and measurement of systolic and diastolic blood pressure. 3/ Regular cycles within the range of 2 days from the average duration 4/ Length of cycles 25 - 35 days. S/ At least one untreated cycle of normal length before the start of treatment. Two different schedules of treatment were used (see below) in two different series of cycles, group (I) and (II). Table I presents the schedules in each group and clinical data *Norethindrone (NET): Sodertalje, Sweden.
446
17_ethinyl-19.nortestosterotl~. SOtng tablets were supplied by AB Astra-Syntex,
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1972
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CONTRACEPTION
concerning the women. In group (I) 82 per cent of the women had been pregnant at least once before (mean 3.0 pregnancies), while in group (II) 79 per cent had been pregnant at least once before (mean 2.75 pregnancies). The mean age was around 30 years. All women
agreed to have a curettage
performed Group
Schedule
should the treatment Group
1
NET 100 mg for 2 days one week before next menstruation (= day 21 22 in a 38-daycycle)
of treatment
fail II
NET 50 mg for 4 days or I day 19 - 21, repeated at 28 days intervals
50
72 (42 from group
Age in years. mean and range
31 (19-43)
30 (19-45)
Previous pregnancies,
123
157
81
79.2
Number
of women
Nos.
Percentage of women with at least one previous pregnancy Table I:
Data concerning group (II).
volunteers
and the schedule
of treatment
I)
in group (I) and
Treatment: A total dose of 200 mg of norethindrone was given orally during each menstural cycle throughout the study. Two different schedules of treatment were used (Table I). In the first series of cycles, group (I), 100 mg was given orally each evening of the 8th and 7th day before the expected start of the next menstruation, e.g. day 21 - 22 in a Z&day-cycle. In the second series, group (II). 50 mg was given on day 19 - 22 in the first
-
I
Group
1 Expected start of menstruation J
NET 100 mg daily 1 J
1 -8
-7
-6
-4
-3
1 St blood sample
2
-1
2nd blood sample
4
NET 50 mg daily j. J1
19
21)
+1
+3
days start
from expected of menstruation
.-
I-
Group
0
II
9 21
22
23
24
25
1st blood sample
26 2nd blood sample
27
30 28 29 days from onset of last bleeding.
L....
Figure
1: Schedule for treatment and blood sampling. In group (II) the 4day-treatment was repeated every 4 weeks after the first treatment cycle.
JUNK 1972
VOL. 5 KO. 6
447
CONTRACEPTION
cycle and then for 4 days every fourth week in a rigid schedule. The two schedules are presented graphically in Figure 1, In a few cycles, the treatment was started one day earlier or later for practical reasons. The treatment was given during 301 cycles, 155 in group (I) and 146 in group (II). The women were usually seen twice every cycle. The first visit took place within three days before starting treatment and the second one about 7 - 10 days later, i.e., around the time of the onset of the vaginal bleeding. The women were interviewed concerning onset of vaginal bleeding, duration and amount of flow, possible discomfort or nausea, etc. Samples: At each visit, two blood samples then frozen until the assays of progesterone,
of 5 ml of venous blood were taken and were estradiol and HCG were performed.
Assay methods Progesterone by Johansson
in plasma (6).
was determined
by the competitive
Estradiol in plasma was determined by radioimmunoassay (7), excluding the thin layer chromatography step (8).
protein
binding
as described
assay described
by Hotchkiss
et al.
The detection of HCG in serum was performed with a radioimmunoassay system using two different antisera for the discrimination between LH and HCG as described by Wide (4).
RESULTS Study A. Effect on the levels of progesterone
and estradiol
The results of the assays of progesterone and estradiol in 10 women during 10 control cycles and 10 treatment cycles are presented in a composite graph in Figure 2, where the cycles have been synchronized to the day before the start of the treatment. The plasma levels of progesterone were drastically reduced as compared to the control cycles in agreement with previous results (1). The plasma levels of estradiol showed a similar pattern, although the decrease was less pronounced. The pattern of progesterone and estradiol was quite similar in all treated cycles despite the large variation in dose and duration of treatment. No calculation of the error was made after day 10 in the treated cycles as 5 women had started to bleed before or on this day and the measurement in three other cycles were at the detection limit of the methods used. In one experiment, the treatment was started in the periovulatory period. Fifty mg of NET was given on day 19 - 22 to a woman who in previous control cycles was found to ovulate at about this time of the cycle. The plasma levels of progesterone increased at the end of the treatment and formed a normal phase pattern (Figure 3).
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CONTRACEPTION
Progeslerone D---O10 normal .-.
cycles
10 NET treated ]
cycles
S.E.M Estradlol 0---o 10 normal -
cycles
10 NET treated 1
cycles
SEM
Star1 of treatment
0 0
2
4
6
8
10
12
0
Days
2
L
6
8
10
12
Days
and estradiol during treatment with norethindrone Figure 2. Plasma levels of progesterone as compared to levels during control cycles. The individual cycles were synchronized on the day before start of treatment (= day 0). The mean dose was 37 mg (range 5 - 100) for a mean of 4.3 days (range 2 - 6).
Study
B. Contraceptive
efficacy
Eighty women were treated with 200 mg of NET during 301 cycles and the effect of the treatment was evaluated according to the effect upon ovulation. control of vaginal bleeding, contraceptive effect and side effects. The two different schedules of treatment (Figure I) were compared. Ovulation The number of ovulatory cycles was calculated from the peripheral plasma levels of progesterone and estradiol. Cycles with levels of progesterone 5 ng/ml or more were regarded as ovulatory (5). In 9 cycles, the plasma levels of progesterone were between 1.2 and 5 ng/ml plasma in the first blood sample with a concomitant plasma level of estradiol exceeding 100 pg/ml plasma. As these are values usually found in the periovulatory period of normal cycles (8), these cycles were also included in the group of ovulatory cycles. Using these criteria, 210 out of 301 cycles were regarded as ovulatory (70%). The rates of ovulatory cycles in group (I) and (II), before and during treatment are presented in Table II.
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14
CONTRACEPTION
4
Progesterone ng/ml prosma
Q---O Oestradiol
ng/lOOmt plasma
A
B-M J
NET 50 mg
;
JW
‘d
12
14
16
18
20
22
24
I
o-d
26
28
30
,P
9,
32
‘.&
34
Day of cycle Figure 3. Plasma levels of progesterone and estradiol during treatment of norethindrone during 4 days, starting in the periovulatory with late ovulation.
with 50 mg daily period in a woman
1
Table I!. Percentage of ovulatory cycles before (= 1st cycles) and during treatment according to the peripheral levels of progesterone and estradiol. Control of vaginal bleeding The mean duration of vaginal bleeding was virtually unchanged by the treatment from 5.5 to 5.1 days in group (I) and from 5.4 to 5.0 days in group (II). The amount of bleeding was the same as before treatment in most cycles as judged by the women. The mean length of the cycles was also almost unaltered, from 29.1 to 28.8 days in group (I) and from 28.9 to 28.8 days in group (II). These data are presented graphically in Figure 4. However, the variation in length of the cycles was larger during treatment than before (Figure 4). This was even more obvious when the number of days from the intake of the last tablet to the day of the start of the vaginal bleeding was recorded (Figure 5). In the majority of the cycles, the vaginal flow started within 3 - 8 days from the last tablet, but in 58 cycles. 22 in
450
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CONTRACEPTION
group (I) and 36 in group (II), this time lag between the end of the treatment and the start of the vaginal flow, was longer (9 - 15 days). To find an explanation for this, the progesterone levels in ovulatory cycles with shorter and longer time lag were compared. Nineteen out of 22 longer cycles in group (I) and 74 out of 36 in group (II) were found to be ovulatory, according to the criteria described above. Figure 6 presents the levels of progesterone in these ovulatory cycles. In the q,cles with a longer time lag between treatment and vaginal bleeding, the levels of progesterone rose from lower values, before the start of the treatment. to values comparable to midluteal phase levels at about the expected start of the menstrual bleeding, whereas, cycles with a shorter time lag showed the opposite pattern with decreasing levels of progesterone.
Gvatial of
vagind bleeding W=
GroupII---
During treatment
60i 50 40 30 20 10
__ __z
__
__.,
I_
IAL!J!3* 3
k!!4_ I--
i--
-_a
L
5
6
7
8
9 ~10
Figure 4. Length of cycles and duration during treatment.
22 25 28 3l U 37 24 27 30 33 36 39
of vaginal bleeding in both groups, before and
Contraceptive effect Twenty-two pregnancies occurred during 301 cycles; IO in group Eighteen of these were recognized by rising levels of progesterone, by clinical signs of pregnancy (amenorrhea, etc.). The remaining 4 group) were recognized only by the detection of HCG. They had the expected time and two of them also had extra bleedings respectively, after the cessation of the 11menstruationI’
(I) and 12 in group (II). estradiol and HCG and pregnancies (2 from each vaginal bleeding at about starting 5 and 8 days,
In the group of 18 pregnancies, recognized not only by the detection of HCG in serum but also by rising levels of progesterone and estradiol and of amenorrhea, curettage was
CONTRACEPTION
Group1
3 L
5
6
7
8
9
10 11 12 13 vi 15
Figure 5. Day of onset of vaginal bleeding calculated was taken (= day 0).
from the day when the last tablet
performed as early as possible. An intrauterine pregnancy was found upon histological examination in all women except one. The trophoblasts appeared to be normal in all specimens. Progesterone, estradiol and HCG were measured weekly and were found to increase within normal levels until the pregnancies were terminated artificially. In one of the women, a decidual reaction of the endometrium was found but no chorion villi could be identified. There were no clinical signs of an extrauterine pregnancy. In 2 out of the 8 “clinical” pregnancies in group (I), HCG was found in the first blood sample which in both cases was taken 8 days before the expected start of the next period, i.e., just before the start of the treatment. These two women were obviously pregnant before the treatment was started. In the remaining 6 pregnancies in group (I) and in IO “clinical” pregnancies in group (II), HCG was found for the first time in the blood sample taken at about the time of the expected menstruation. Two of these 16 pregnant women had periovulatory levels (see above) of progesterone and estradiol in the first blood sample whereas the other 14 had postovulatory levels. Side effects Nausea occurred in 18 cycles in group (I) and in I6 cycles in group (II) (total, I1 per cent of 301 cycles). The nausea was usually mild and occurred during the first morning hours on the day after the first dose of the drug. A few women experienced a more pronounced nausea and one woman from each group discontinued treatment for this reason. Slight inter-menstrual bleeding for 1 or 2 days occurred in 6 and 4 cycles, respectively (total 3.3%). One woman in group (II) had a mild mental depression and discontinued the treatment. 452
JUNE
1972
VOL.
5 NO.
6
CONTRACEPTION
Time
GrOUpi
lag n93 3-8 DcVs
f.
nll
4, ‘\
Mean5.E.M.
Group11
I n56
153-9 Days
n2.4
I., \
\
\
\
10. , n2L
59-15
f’*
,’
/ X
\c ./%\
.4
/
.’ \ ‘\
.,
nL6 ‘E
OJ
I st
2nd blood
Mood sample f Start of treatment Figure 6.
Yample Expected start of bleeding
Plasma levels of progesterone in ovulatory cycles of both groups with shorter (3 - 8 days) and longer (9 - 15 days) time lag between end of treatment and start of vaginal bleeding.
One woman in group (II) with acne vulgaris before treatment found that her condition impaired by the treatment and, therefore stopped taking the tablets.
was
Two women reported weight gain. No other side effects were found or reported by the women. Discontinuation
of treatment
women from group (I) and 6 from group (II) discontinued following reasons:
Three
Group I: one because of nausea, two for non-medical reasons. Group II: one because ot’ nausea, oue because of a mild mental impairment of acne vulgaris and three for non-medical reasons.
the treatment
for the
depression,
one for
CONTRACEPTION
DJSCUSSION The drastic decrease of the peripheral levels of progesterone after postovulatory administration of NET, found in this study is in agreement with findings previously reported (1). Preliminary reports of a similar, but less drastic effect upon the peripheral levels of estradiol (2. 3) were verified (Figure 2). The mechanism through which NET exerts its effect is not known. The depressed peripheral levels of progesterone probably reflect a diminished production of this hormone from the corpus luteum (1). Aakvaag (9) found that chlomradinone acetate, another synthetic gestagen, inhibited the conversion of pregnenolone to progesterone in porcine ovaries i,r vitro and Mukhejee et al. ( 10) found the same effect of norgestrel, still another synthetic gestagen. upon the function of human corpora lutea. However, injections of HCG (1) or luteinizing hormone (Johansson, to be published), during treatment, reversed the effect of norethindrone as reflected by increasing plasma levels of progesterone and estradiol. It is possible. therefore, that the effect of postovulatory administration of NET is a direct inhibition of the steroidogenesis in the corpus luteum, but it might also be a negative feed back on the hypothalamic-pituitary-system. Injections of 6.000 - 9,000 IU of HCG did not restore the plasma levels of progesterone to normal luteal phase levels when a total dose of 200 mg of NET or more was used (I ). This dose was therefore chosen to test the contraceptive efficacy of the treatment. Our hypothesis for the contraceptive effect of this treatment was that the depressed levels of progesterone, and to a lesser extent of estradiol, would change the development of the postovulatory endometrium. A prerequisite would be that the drug itself did not substitute for the low levels of progesterone. NET is inactive in the pregnancy maintenance test in rats (1 1). which might support the possibility that the drug would not substitute for the depressed levels of progesterone. A contraceptive effect would then be achieved through impaired possibilities for normal implantation. The treatment would be “menses-inducing” Twenty-two pregnancies occurred during 301 cycles in 80 women. Four of these pregnancies were detected only by the presence of HCG in serum. It is difficult to ascertain whether this was an effect of the treatment, but it is not unlikely that these 4 cases were very early spontaneous abortions. They represented 18 per cent of the total number of pregnancies, a figure that one might expect to get in a study like this where a very sensitive pregnancy test was used. Eighteen pregnant women did not abort. Two of them did not take their tablets as directed (one took half the dose, the other started her treatment one week too late). Twenty patients took the tablets on schedule. However, 4 of these aborted within 8 days of the expected time of menstruation. There was no substantial difference concerning contraceptive efficacy, rate of ovulation, control of vaginal bleeding or side effects between the two different dose schedules. To be able to interpret this result correctly, one would have to know how many pregnancies would have occurred if no contraceptive method had been used. Eighty per cent of the women were of proven fertility. At least 70 per cent of the cycles were found to be ovulatory. As the blood samples were taken with rather long intervals and with occasiona omittance of blood samples, this estimation is probably too low. According to Peel & Potts (12) the percentage of married women who will be pregnant after one month
454
JUNE 1972
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CONTRACEPTION
of unprotected coitus is about 35 per cent, after 2 months about 48 per cent and after 3 months about 58 per cent. Using these figures and assuming that the women in this study were as fertile as the women in the report by Peel and Potts, the expected number of pregnancies would be about 64. If only those women with proven fertility and only the ovulatory cycles in this study are used for the calculation, the expected number of pregnancies would be about 36. The difference between the expected number of pregnancies without treatment and the actual number of pregnancies is considerable and might justify the assumption that the treatment had a certain contraceptive effect. The findings seem to support the proposition that the treatment had an effect only when given well before implantation and if the blastocyst was implanted, the pregnancy developed normally. When abortion did not occur, piogesterone, estradiol and HCG increased within normal limits until the pregnancy was terminated artificially. The very early abortions represented only 18 per ceni of the total number of pregnancies in the study, suggesting that they were spontaneous abortions and not :I result of a pathological implantation caused by the treatment. Finally, the histological examination of the artificially aborted pregnancies did not reveal extensive necrosis or other abnormalities. Neither the mean duration of the vaginal bleedings nor the amount of flow was significantly altered, but some of the cycles had a longer time lag between the end of treatment and the start of vaginal bleeding than the majority of cycles. Most of these cycles were ovulatory. As judged by the progesterone levels, presented in Figure 6, ovulation occurred later in these cycles as compared with cycles with a shorter time lag. Therefore. in many of the ” long time lag cycles”. treatment was started quite near the time of ovulation. It seems that the treatment in these cycles was not able to depress the levels of progesterone and estradiol during mid- and late-lutheal phases as illustrated in the cycle presented in Figure 3. One might suspect. that most of the pregnancies would have occurred in such cycles, but only 2 of them actually did. Considering this fact, together with detection of HCG even before the start of treatment in 2 of the pregnancies, it would seem advisable to start the treatment somewhat earlier in the cycle, e.g., on day I6 or 17 and to continue until day 2. The low contraceptive effect found for NET in the present study obviously does not merit further use. However, a schedule of treatment aimed to cover the period between ovulation and implantation, as suggested above, might ha+, a better contraceptive efficacy and is presently being studied.
ACKNOWLEDGEMENTS This study was supported by the Population Council, New York, N.Y., the Swedish Medical Research Council, project No. 17X -3495 and 13X-3 14.5 and the Ford Foundation, New York, N.Y. The authors are indebted to their technical staff for skilled assistance.
CONTRACEPTION
REFERENCES I. Johansson, E.D.B.: Depression of the progesterone levels in women synthetic gestagens after ovulation. Acta endocr. (Kbh.) 68:779 (1971).
treated
with
2. Johansson, E.D.B.: Studies of compounds that interfere with the normal function of the corpus luteum in women. VIIth World Congress on Fertility and Sterility, Tokyo (1971). 3. Nygren, K.G. and Johansson, E.D.B.: Interference with the normal function of the corpus luteum by oral administration of synthetic gestagens. Acta endocr. (Kbh.) Suppl. 155, 170 (1971). 4. Wide, L.: Early diagnoses of pregnancy.
Lancet, 2: 863 (1969).
5. Johansson, E.D.B., Wide, L. and Gemzell, progesterone in plasma and LH and oestrogens cycles. Acta endocr. (Kbh.) 68:502 (197 1). 6. Johansson, E.D.B.: A simplified (Kbh.) Suppl. 147, 188 (1970).
procedure
C.: Luteinizing hormone (LH) and in urine during 42 normal menstrual
for the assay of progesterone.
Acta endocr.
7. Hotchkiss, J., Atkinson, L.E. and Knobil, E.: Time course of serum estrogen and luteinizing hormone (LH) concentrations during the menstrual cycle of the rhesus monkey. Endocrinology 89:177 (1971). 8. Edqvist, L.E. and Johansson, E.D.B.: Radioimmunoassay of estrone and estradiol human and bovine peripheral plasma. Acta endocr. (Kbh.) in press (1972). 9. Aakvaag, A.: Steroid formation 65:261 (1970).
in porcine
ovarian tissue in vitro. Acta endocr.
in
(Kbh.)
IO. Mukherjee, T.K., Wright, S.W., Davidson, N.J.H. and Fotherby, K.: Effect of norgestrel on corpus luteum function. J. Obstet. Gynaec. Brit. Commwth 79:175 (1972). 1 I. Edgren, R.A. in Contraception: The chemical control M. Dekker Inc., New York. (1969). 12. Peel, _I. and Potts, M. Textbook (1969).
456
of contraceptive
of fertility.
practice.
D. Lednicer, Editor,
Cambridge
JUNE 1972
University
Press
VOL. 5 NO. 6
CONTRACEPTION IN WOMEN WITH LARGE DOSES OF NORETHINDRONE
KarlCGsta
Nygren, Elof D.B. Johansson
The Department of Obstetrics and Gynecology University Hospital, Uppsala, Sweden
and Leif Wide
and the Department
of Clinical Chemistry,
ABSTRACT The postovulatory administration of large doses of norethindrone to women has previously been shown to drastically reduce the peripheral plasma levels of progesterone. In IO treatment cycles, compared to 10 control cycles from the same women, this effect was confirmed and a similar effect upon the peripheral plasma levels of estradiol was found. The contraceptive efficacy of this treatment was tested. Eighty volunteers were treated during 301 cycles. Eighty per cent of the women had been pregnant before. The plasma levels of progesterone and estradiol were determined before and after treatment in each cycle. A very sensitive test for the detection in serum of human chorionic gonadotrophin (HCG) was included. A total dose of 200 mg of norethindrone was used in two different schedules (100 mg daily on day 21 - 22 and 50 mg daily on day 19 - 22). Twenty-two pregnancies occurred, but in 4 of these women, the only indication of pregnancy was the detection of HCG. Two women did not take the tablets according to schedule, leaving 16 patients who had to be aborted therapeutically. At least 70 per cent of the cycles were found to be ovulatory. Control of vaginal bleeding was good and only minor side effects occurred. The treatment possibly reduced fertility in this group of women but the contraceptive efficacy was obviously too low to merit further use of the present dose and schedules.
Accepted
JUNE
1972
for
publication
A70L.
3; NO.
May 8,
6
1972.
445
CONTRACEPTION
INTRODUCTION Norethindrone (NET)* and other synthetic gestagens have been shown to drastically reduce the peripheral plasma levels of progesterone when given after ovulation to women (1). In preliminary reports (2, 3), postovulatory treatment with NET has also been claimed to depress the peripheral levels of estradiol. To verify this, an investigation was made concerning the effect of postovulatory treatment with NET upon the peripheral levels of both progesterone and estradiol. It was considered that the decreased periphera1 levels of the ovarian steroids might influence the development of the postovulatory endometrium and thereby impair the possibilities for normal implantation of the blastocyst. To investigate the contraceptive efficacy of such a treatment, NET was given postovulatory to pregnancy exposed women. As this treatment might result in a ” menstrual bleeding” that would, in fact, be an early abortion, a very sensitive test for the detection in serum of human chorionic gonadotrophin (HCG) (4) was included in the study. METHODS Study
A. Effect on the levels of progesterone
and estradiol
Subjects: Ten healthy women, 20 to 32 years of age, all employed volunteered for this study. All had regular menstrual cycles. Treatment: Various doses number of days (mean postovulatory day. The progesterone and estradiol
by the hospital,
of NET (mean 37 mg, range 5 100 mg) were given for various 4.3 days, range 2 6 days) starting on the third or fourth day of ovulation was calculated from the plasma levels of as described in previous papers ( 1, 5 ).
Samples: Daily blood samples for the assay of progesterone and estradiol veni puncture during the periovulatory and luteal phases of each cycle. Study
B. Contraceptive
were collected
by
efficacy
Subjects: Women seeking ccntraceptive advice at the University Hospital of Uppsala. Sweden, volunteered for this investigation. Those fulfilling the following criteria were accepted: I / Age 18 45 years. 2/ Good general health and normal findings upon gynecological examination, including a vaginal smear, palpation of the mammae and measurement of systolic and diastolic blood pressure. 3/ Regular cycles within the range of 2 days from the average duration 4/ Length of cycles 25 - 35 days. S/ At least one untreated cycle of normal length before the start of treatment. Two different schedules of treatment were used (see below) in two different series of cycles, group (I) and (II). Table I presents the schedules in each group and clinical data *Norethindrone (NET): Sodertalje, Sweden.
446
17_ethinyl-19.nortestosterotl~. SOtng tablets were supplied by AB Astra-Syntex,
JUNE
1972
VOL.
5 NO. 6
CONTRACEPTION
concerning the women. In group (I) 82 per cent of the women had been pregnant at least once before (mean 3.0 pregnancies), while in group (II) 79 per cent had been pregnant at least once before (mean 2.75 pregnancies). The mean age was around 30 years. All women
agreed to have a curettage
performed Group
Schedule
should the treatment Group
1
NET 100 mg for 2 days one week before next menstruation (= day 21 22 in a 38-daycycle)
of treatment
fail II
NET 50 mg for 4 days or I day 19 - 21, repeated at 28 days intervals
50
72 (42 from group
Age in years. mean and range
31 (19-43)
30 (19-45)
Previous pregnancies,
123
157
81
79.2
Number
of women
Nos.
Percentage of women with at least one previous pregnancy Table I:
Data concerning group (II).
volunteers
and the schedule
of treatment
I)
in group (I) and
Treatment: A total dose of 200 mg of norethindrone was given orally during each menstural cycle throughout the study. Two different schedules of treatment were used (Table I). In the first series of cycles, group (I), 100 mg was given orally each evening of the 8th and 7th day before the expected start of the next menstruation, e.g. day 21 - 22 in a Z&day-cycle. In the second series, group (II). 50 mg was given on day 19 - 22 in the first
-
I
Group
1 Expected start of menstruation J
NET 100 mg daily 1 J
1 -8
-7
-6
-4
-3
1 St blood sample
2
-1
2nd blood sample
4
NET 50 mg daily j. J1
19
21)
+1
+3
days start
from expected of menstruation
.-
I-
Group
0
II
9 21
22
23
24
25
1st blood sample
26 2nd blood sample
27
30 28 29 days from onset of last bleeding.
L....
Figure
1: Schedule for treatment and blood sampling. In group (II) the 4day-treatment was repeated every 4 weeks after the first treatment cycle.
JUNK 1972
VOL. 5 KO. 6
447
CONTRACEPTION
cycle and then for 4 days every fourth week in a rigid schedule. The two schedules are presented graphically in Figure 1, In a few cycles, the treatment was started one day earlier or later for practical reasons. The treatment was given during 301 cycles, 155 in group (I) and 146 in group (II). The women were usually seen twice every cycle. The first visit took place within three days before starting treatment and the second one about 7 - 10 days later, i.e., around the time of the onset of the vaginal bleeding. The women were interviewed concerning onset of vaginal bleeding, duration and amount of flow, possible discomfort or nausea, etc. Samples: At each visit, two blood samples then frozen until the assays of progesterone,
of 5 ml of venous blood were taken and were estradiol and HCG were performed.
Assay methods Progesterone by Johansson
in plasma (6).
was determined
by the competitive
Estradiol in plasma was determined by radioimmunoassay (7), excluding the thin layer chromatography step (8).
protein
binding
as described
assay described
by Hotchkiss
et al.
The detection of HCG in serum was performed with a radioimmunoassay system using two different antisera for the discrimination between LH and HCG as described by Wide (4).
RESULTS Study A. Effect on the levels of progesterone
and estradiol
The results of the assays of progesterone and estradiol in 10 women during 10 control cycles and 10 treatment cycles are presented in a composite graph in Figure 2, where the cycles have been synchronized to the day before the start of the treatment. The plasma levels of progesterone were drastically reduced as compared to the control cycles in agreement with previous results (1). The plasma levels of estradiol showed a similar pattern, although the decrease was less pronounced. The pattern of progesterone and estradiol was quite similar in all treated cycles despite the large variation in dose and duration of treatment. No calculation of the error was made after day 10 in the treated cycles as 5 women had started to bleed before or on this day and the measurement in three other cycles were at the detection limit of the methods used. In one experiment, the treatment was started in the periovulatory period. Fifty mg of NET was given on day 19 - 22 to a woman who in previous control cycles was found to ovulate at about this time of the cycle. The plasma levels of progesterone increased at the end of the treatment and formed a normal phase pattern (Figure 3).
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CONTRACEPTION
Progeslerone D---O10 normal .-.
cycles
10 NET treated ]
cycles
S.E.M Estradlol 0---o 10 normal -
cycles
10 NET treated 1
cycles
SEM
Star1 of treatment
0 0
2
4
6
8
10
12
0
Days
2
L
6
8
10
12
Days
and estradiol during treatment with norethindrone Figure 2. Plasma levels of progesterone as compared to levels during control cycles. The individual cycles were synchronized on the day before start of treatment (= day 0). The mean dose was 37 mg (range 5 - 100) for a mean of 4.3 days (range 2 - 6).
Study
B. Contraceptive
efficacy
Eighty women were treated with 200 mg of NET during 301 cycles and the effect of the treatment was evaluated according to the effect upon ovulation. control of vaginal bleeding, contraceptive effect and side effects. The two different schedules of treatment (Figure I) were compared. Ovulation The number of ovulatory cycles was calculated from the peripheral plasma levels of progesterone and estradiol. Cycles with levels of progesterone 5 ng/ml or more were regarded as ovulatory (5). In 9 cycles, the plasma levels of progesterone were between 1.2 and 5 ng/ml plasma in the first blood sample with a concomitant plasma level of estradiol exceeding 100 pg/ml plasma. As these are values usually found in the periovulatory period of normal cycles (8), these cycles were also included in the group of ovulatory cycles. Using these criteria, 210 out of 301 cycles were regarded as ovulatory (70%). The rates of ovulatory cycles in group (I) and (II), before and during treatment are presented in Table II.
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14
CONTRACEPTION
4
Progesterone ng/ml prosma
Q---O Oestradiol
ng/lOOmt plasma
A
B-M J
NET 50 mg
;
JW
‘d
12
14
16
18
20
22
24
I
o-d
26
28
30
,P
9,
32
‘.&
34
Day of cycle Figure 3. Plasma levels of progesterone and estradiol during treatment of norethindrone during 4 days, starting in the periovulatory with late ovulation.
with 50 mg daily period in a woman
1
Table I!. Percentage of ovulatory cycles before (= 1st cycles) and during treatment according to the peripheral levels of progesterone and estradiol. Control of vaginal bleeding The mean duration of vaginal bleeding was virtually unchanged by the treatment from 5.5 to 5.1 days in group (I) and from 5.4 to 5.0 days in group (II). The amount of bleeding was the same as before treatment in most cycles as judged by the women. The mean length of the cycles was also almost unaltered, from 29.1 to 28.8 days in group (I) and from 28.9 to 28.8 days in group (II). These data are presented graphically in Figure 4. However, the variation in length of the cycles was larger during treatment than before (Figure 4). This was even more obvious when the number of days from the intake of the last tablet to the day of the start of the vaginal bleeding was recorded (Figure 5). In the majority of the cycles, the vaginal flow started within 3 - 8 days from the last tablet, but in 58 cycles. 22 in
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group (I) and 36 in group (II), this time lag between the end of the treatment and the start of the vaginal flow, was longer (9 - 15 days). To find an explanation for this, the progesterone levels in ovulatory cycles with shorter and longer time lag were compared. Nineteen out of 22 longer cycles in group (I) and 74 out of 36 in group (II) were found to be ovulatory, according to the criteria described above. Figure 6 presents the levels of progesterone in these ovulatory cycles. In the q,cles with a longer time lag between treatment and vaginal bleeding, the levels of progesterone rose from lower values, before the start of the treatment. to values comparable to midluteal phase levels at about the expected start of the menstrual bleeding, whereas, cycles with a shorter time lag showed the opposite pattern with decreasing levels of progesterone.
Gvatial of
vagind bleeding W=
GroupII---
During treatment
60i 50 40 30 20 10
__ __z
__
__.,
I_
IAL!J!3* 3
k!!4_ I--
i--
-_a
L
5
6
7
8
9 ~10
Figure 4. Length of cycles and duration during treatment.
22 25 28 3l U 37 24 27 30 33 36 39
of vaginal bleeding in both groups, before and
Contraceptive effect Twenty-two pregnancies occurred during 301 cycles; IO in group Eighteen of these were recognized by rising levels of progesterone, by clinical signs of pregnancy (amenorrhea, etc.). The remaining 4 group) were recognized only by the detection of HCG. They had the expected time and two of them also had extra bleedings respectively, after the cessation of the 11menstruationI’
(I) and 12 in group (II). estradiol and HCG and pregnancies (2 from each vaginal bleeding at about starting 5 and 8 days,
In the group of 18 pregnancies, recognized not only by the detection of HCG in serum but also by rising levels of progesterone and estradiol and of amenorrhea, curettage was
CONTRACEPTION
Group1
3 L
5
6
7
8
9
10 11 12 13 vi 15
Figure 5. Day of onset of vaginal bleeding calculated was taken (= day 0).
from the day when the last tablet
performed as early as possible. An intrauterine pregnancy was found upon histological examination in all women except one. The trophoblasts appeared to be normal in all specimens. Progesterone, estradiol and HCG were measured weekly and were found to increase within normal levels until the pregnancies were terminated artificially. In one of the women, a decidual reaction of the endometrium was found but no chorion villi could be identified. There were no clinical signs of an extrauterine pregnancy. In 2 out of the 8 “clinical” pregnancies in group (I), HCG was found in the first blood sample which in both cases was taken 8 days before the expected start of the next period, i.e., just before the start of the treatment. These two women were obviously pregnant before the treatment was started. In the remaining 6 pregnancies in group (I) and in IO “clinical” pregnancies in group (II), HCG was found for the first time in the blood sample taken at about the time of the expected menstruation. Two of these 16 pregnant women had periovulatory levels (see above) of progesterone and estradiol in the first blood sample whereas the other 14 had postovulatory levels. Side effects Nausea occurred in 18 cycles in group (I) and in I6 cycles in group (II) (total, I1 per cent of 301 cycles). The nausea was usually mild and occurred during the first morning hours on the day after the first dose of the drug. A few women experienced a more pronounced nausea and one woman from each group discontinued treatment for this reason. Slight inter-menstrual bleeding for 1 or 2 days occurred in 6 and 4 cycles, respectively (total 3.3%). One woman in group (II) had a mild mental depression and discontinued the treatment. 452
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1972
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Time
GrOUpi
lag n93 3-8 DcVs
f.
nll
4, ‘\
Mean5.E.M.
Group11
I n56
153-9 Days
n2.4
I., \
\
\
\
10. , n2L
59-15
f’*
,’
/ X
\c ./%\
.4
/
.’ \ ‘\
.,
nL6 ‘E
OJ
I st
2nd blood
Mood sample f Start of treatment Figure 6.
Yample Expected start of bleeding
Plasma levels of progesterone in ovulatory cycles of both groups with shorter (3 - 8 days) and longer (9 - 15 days) time lag between end of treatment and start of vaginal bleeding.
One woman in group (II) with acne vulgaris before treatment found that her condition impaired by the treatment and, therefore stopped taking the tablets.
was
Two women reported weight gain. No other side effects were found or reported by the women. Discontinuation
of treatment
women from group (I) and 6 from group (II) discontinued following reasons:
Three
Group I: one because of nausea, two for non-medical reasons. Group II: one because ot’ nausea, oue because of a mild mental impairment of acne vulgaris and three for non-medical reasons.
the treatment
for the
depression,
one for
CONTRACEPTION
DJSCUSSION The drastic decrease of the peripheral levels of progesterone after postovulatory administration of NET, found in this study is in agreement with findings previously reported (1). Preliminary reports of a similar, but less drastic effect upon the peripheral levels of estradiol (2. 3) were verified (Figure 2). The mechanism through which NET exerts its effect is not known. The depressed peripheral levels of progesterone probably reflect a diminished production of this hormone from the corpus luteum (1). Aakvaag (9) found that chlomradinone acetate, another synthetic gestagen, inhibited the conversion of pregnenolone to progesterone in porcine ovaries i,r vitro and Mukhejee et al. ( 10) found the same effect of norgestrel, still another synthetic gestagen. upon the function of human corpora lutea. However, injections of HCG (1) or luteinizing hormone (Johansson, to be published), during treatment, reversed the effect of norethindrone as reflected by increasing plasma levels of progesterone and estradiol. It is possible. therefore, that the effect of postovulatory administration of NET is a direct inhibition of the steroidogenesis in the corpus luteum, but it might also be a negative feed back on the hypothalamic-pituitary-system. Injections of 6.000 - 9,000 IU of HCG did not restore the plasma levels of progesterone to normal luteal phase levels when a total dose of 200 mg of NET or more was used (I ). This dose was therefore chosen to test the contraceptive efficacy of the treatment. Our hypothesis for the contraceptive effect of this treatment was that the depressed levels of progesterone, and to a lesser extent of estradiol, would change the development of the postovulatory endometrium. A prerequisite would be that the drug itself did not substitute for the low levels of progesterone. NET is inactive in the pregnancy maintenance test in rats (1 1). which might support the possibility that the drug would not substitute for the depressed levels of progesterone. A contraceptive effect would then be achieved through impaired possibilities for normal implantation. The treatment would be “menses-inducing” Twenty-two pregnancies occurred during 301 cycles in 80 women. Four of these pregnancies were detected only by the presence of HCG in serum. It is difficult to ascertain whether this was an effect of the treatment, but it is not unlikely that these 4 cases were very early spontaneous abortions. They represented 18 per cent of the total number of pregnancies, a figure that one might expect to get in a study like this where a very sensitive pregnancy test was used. Eighteen pregnant women did not abort. Two of them did not take their tablets as directed (one took half the dose, the other started her treatment one week too late). Twenty patients took the tablets on schedule. However, 4 of these aborted within 8 days of the expected time of menstruation. There was no substantial difference concerning contraceptive efficacy, rate of ovulation, control of vaginal bleeding or side effects between the two different dose schedules. To be able to interpret this result correctly, one would have to know how many pregnancies would have occurred if no contraceptive method had been used. Eighty per cent of the women were of proven fertility. At least 70 per cent of the cycles were found to be ovulatory. As the blood samples were taken with rather long intervals and with occasiona omittance of blood samples, this estimation is probably too low. According to Peel & Potts (12) the percentage of married women who will be pregnant after one month
454
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of unprotected coitus is about 35 per cent, after 2 months about 48 per cent and after 3 months about 58 per cent. Using these figures and assuming that the women in this study were as fertile as the women in the report by Peel and Potts, the expected number of pregnancies would be about 64. If only those women with proven fertility and only the ovulatory cycles in this study are used for the calculation, the expected number of pregnancies would be about 36. The difference between the expected number of pregnancies without treatment and the actual number of pregnancies is considerable and might justify the assumption that the treatment had a certain contraceptive effect. The findings seem to support the proposition that the treatment had an effect only when given well before implantation and if the blastocyst was implanted, the pregnancy developed normally. When abortion did not occur, piogesterone, estradiol and HCG increased within normal limits until the pregnancy was terminated artificially. The very early abortions represented only 18 per ceni of the total number of pregnancies in the study, suggesting that they were spontaneous abortions and not :I result of a pathological implantation caused by the treatment. Finally, the histological examination of the artificially aborted pregnancies did not reveal extensive necrosis or other abnormalities. Neither the mean duration of the vaginal bleedings nor the amount of flow was significantly altered, but some of the cycles had a longer time lag between the end of treatment and the start of vaginal bleeding than the majority of cycles. Most of these cycles were ovulatory. As judged by the progesterone levels, presented in Figure 6, ovulation occurred later in these cycles as compared with cycles with a shorter time lag. Therefore. in many of the ” long time lag cycles”. treatment was started quite near the time of ovulation. It seems that the treatment in these cycles was not able to depress the levels of progesterone and estradiol during mid- and late-lutheal phases as illustrated in the cycle presented in Figure 3. One might suspect. that most of the pregnancies would have occurred in such cycles, but only 2 of them actually did. Considering this fact, together with detection of HCG even before the start of treatment in 2 of the pregnancies, it would seem advisable to start the treatment somewhat earlier in the cycle, e.g., on day I6 or 17 and to continue until day 2. The low contraceptive effect found for NET in the present study obviously does not merit further use. However, a schedule of treatment aimed to cover the period between ovulation and implantation, as suggested above, might ha+, a better contraceptive efficacy and is presently being studied.
ACKNOWLEDGEMENTS This study was supported by the Population Council, New York, N.Y., the Swedish Medical Research Council, project No. 17X -3495 and 13X-3 14.5 and the Ford Foundation, New York, N.Y. The authors are indebted to their technical staff for skilled assistance.
CONTRACEPTION
REFERENCES I. Johansson, E.D.B.: Depression of the progesterone levels in women synthetic gestagens after ovulation. Acta endocr. (Kbh.) 68:779 (1971).
treated
with
2. Johansson, E.D.B.: Studies of compounds that interfere with the normal function of the corpus luteum in women. VIIth World Congress on Fertility and Sterility, Tokyo (1971). 3. Nygren, K.G. and Johansson, E.D.B.: Interference with the normal function of the corpus luteum by oral administration of synthetic gestagens. Acta endocr. (Kbh.) Suppl. 155, 170 (1971). 4. Wide, L.: Early diagnoses of pregnancy.
Lancet, 2: 863 (1969).
5. Johansson, E.D.B., Wide, L. and Gemzell, progesterone in plasma and LH and oestrogens cycles. Acta endocr. (Kbh.) 68:502 (197 1). 6. Johansson, E.D.B.: A simplified (Kbh.) Suppl. 147, 188 (1970).
procedure
C.: Luteinizing hormone (LH) and in urine during 42 normal menstrual
for the assay of progesterone.
Acta endocr.
7. Hotchkiss, J., Atkinson, L.E. and Knobil, E.: Time course of serum estrogen and luteinizing hormone (LH) concentrations during the menstrual cycle of the rhesus monkey. Endocrinology 89:177 (1971). 8. Edqvist, L.E. and Johansson, E.D.B.: Radioimmunoassay of estrone and estradiol human and bovine peripheral plasma. Acta endocr. (Kbh.) in press (1972). 9. Aakvaag, A.: Steroid formation 65:261 (1970).
in porcine
ovarian tissue in vitro. Acta endocr.
in
(Kbh.)
IO. Mukherjee, T.K., Wright, S.W., Davidson, N.J.H. and Fotherby, K.: Effect of norgestrel on corpus luteum function. J. Obstet. Gynaec. Brit. Commwth 79:175 (1972). 1 I. Edgren, R.A. in Contraception: The chemical control M. Dekker Inc., New York. (1969). 12. Peel, _I. and Potts, M. Textbook (1969).
456
of contraceptive
of fertility.
practice.
D. Lednicer, Editor,
Cambridge
JUNE 1972
University
Press
VOL. 5 NO. 6