0021-972X/92/7401-0108$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 1 Printed
in U.S.A.
Melatonin and Melatonin-Progestin Combinations Alter Pituitary-Ovarian Function in Women and Can Inhibit Ovulation BETTIE C. G. VOORDOUW, ROBERT EUSER, RENE E. R. VERDONK, BERT TH. ALBERDA, FRANK H. DE JONG, AAT C. DROGENDIJK, BART C. J. M. FAUSER, AND MICHAEL COHEN Center for Reproductive Medicine and Breast Cancer Prevention and Departments of Gynecology and Obstetrics (B.C.G. V., B. Th.A., A.C.D., B.C. J.M.F.) and Medicine (F.H. J.), Dijkzigt University Hospital, Rotterdam; the Department of Gynecology and Obstetrics, Academic Hospital (R.E.), Utrecht; and AMR Pharm Holland B. V. (R.E.R. V., M.C.), The Hague, The Netherlands
ABSTRACT. Although melatonin (MEL) controls seasonal reproductive cyclicity in some mammalian species, its role in women is controversial. In this study data are presented related to the influence of MEL or MEL-progestin combinations on the pituitary-ovarian axis and ovulation in 32 women. MEL was administered in a dosage of 300 mg to 12 women for 4 months [to 8 women daily (days l-30) and to 4 women on days 5-17 of the cycle]. MEL was also combined with the synthetic progestin norethisterone (NET) in an attempt to evaluate MEL’s effect on a partially suppressed pituitary-ovarian axis. In 16 women, 4 combinations were tested on 4 women each on davs 1-21: dosages of 300 mg MEL/O.75 mg NET, 75 mg MEL/O.75 mg NET, -7.5 ma MEL/O.75 ma NET. and 75 me MEL/O.30 me NET. In addition, 2 womelwere medicated wgh 300 mg MELalone, and 2 were medicated with 300 mg MEL/O.15 mg NET on days l21 for 2 months. During the study, LH, FSH, estradiol (E,), and progesterone (PJ blood levels were determined at regular intervals. After a period of 4 months, daily administration of 300 mg
T
HE EXACT functions and role of the pineal gland and its major secretory product melatonin (MEL) are still controversial (1). The most persistent and convincing evidence, however, ascribes the pineal gland a role in regulating reproductive events (2). In 1899, Huebner first associated human precocious puberty with various tumours in the pineal region (3). Subsequent evidence for a pineal role in the regulation of reproductive events linked light exposure and pinealectomy with increased ovarian function in the rat (4, 5). The identification of MEL (6) and the effects of environmental light on its biosynthesis (7) suggested MEL to be the link between light-dark periods and reproductive events. The
MEL (days l-30) caused significantly decreased mean LH levels compared to those in 8 nonmedicated controls (P < 0.001). Also compared to nonmedicated control data, a significant inhibition of P, in the first and fourth medication months (P < 0.001) was observed. LH and E, inhibition reached sianificance in the fourth medication month (P c 0.005). Also, thetreatments of 300 mg MEL (days 5-17) and 75 mg MEL combined with 0.3 mg NET caused a significant decrease in LH, EP, and P, levels compared to those in the nonmedicated control group in the first and fourth medication months (P < 0.05). The data further suggest an additive or synergistic effect between MEL and NET. The medications did not alter sleep-wake rhythms and were not complicated by any side-effects. The presented data suggest that MEL and MEL/NET combinations inhibit ovarian function in women, and that MEL/NET combinations can emerge as effective oral contraceptives. (J Ctin Endocrirwl Metab 74: 108-117, 1992)
inhibitory effects of exogenous MEL on ovarian function in several rodent species further support the hypothesis of pineal regulation of reproductive events (8, 9). In contrast with the situation in small rodents, MEL stimulates gonadal function in sheep. In sheep, elevated serum MEL levels are associated with decreasing day length in the fall and introduce the fertile season (10). Stimulatory and inhibitory effects of MEL in sheep and rodents only become apparent after a significant lag time. In addition, the response is complicated by a refractoriness of the hypothalamic-pituitary-gonadal axis to MEL, which develops over time (11). In humans, only scant data exist on the role of MEL in the regulation of reproductive functions. Endogenous MEL secretion follows the light-dark circadian rhythm, with peak nighttime serum levels, and high intensity light can suppress MEL synthesis (12). Whether environmental light or changes in serum MEL levels influ-
Received August 30, 1990. Address all correspondence and requests for reprints to: Mrs. A. C. G. Voordouw, M.D., Center for Reproductive Medicine and Breast Cancer Prevention, Parklaan 12, 3016 BB Rotterdam, The Netherlands. 108
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 October 2015. at 14:40 For personal use only. No other uses without permission. . All rights reserved.
MEL, PROGESTIN,
AND
PITUITARY-OVARIAN
ence reproductive functions in man is uncertain, although a seasonal influence on the ovulatory LH surge has been described (13). Moreover, it is of interest that conception of dizygotic twin pregnancies is more common in the summer months at the time of continuous light in Finland (14), and in primary hypothalamic amenorrhea high nighttime values of MEL have been found (15). The observed negative correlation between MEL secretion and sexual maturation is also consistent with the hypothesis that MEL is antigonadal in man (16). As part of an ongoing program for the development of an estrogen-free MEL-containing oral contraceptive, this study describes the effects of exogenous MEL alone or combined with the synthetic progestin norethisterone (NET) on pituitary-ovarian function in women.
Subjects
and Methods
Participants
The clinical trials were designedin accordancewith WHO guidelinesfor phaseI and II clinical trials in the development of steroidal contraceptives (17). Thirty-two women received medication for 2-4 months, and eight nonmedicated controls were evaluated for one cycle. The studieswere approved by the institutional review boards for experimentation on humans.A detailed informed consentwas obtained from the experimental subjects.The entire study population was recruited by advertisements, and subjectswere assignedrandomly to the study groups.The pharmaceutical combinationsused were prepared in compliancewith pharmacopoeiaguidesand were tested for bioavailability in uiuo. Inclusion criteria required an age between 18-37 yr, a history of regular menstrual cycles between 22-34 days, a biphasic basal body temperature (BBT) in a prestudy control cycle, a normal menstrual pattern of 3-7 bleeding days, and good general health, as determined by a normal medical history, physical examination, normal electrocardiogram,normal standard hematology (Hb, Ht, erythrocyte sedimentation rate, mean cellular volume, mean cellular hemoglobin, meancellular hemoglobinconcentration, and differentiation), biochemistry (sodium, potassium, creatinin, urea, bilirubin, alkaline phosphatase,SGOT, SGPT, lactate dehydrogenase,rGT, total protein, triglycerides, high and low density lipoproteins, total cholesterol, glucose,total Tq, and free
T,) and urine evaluation (pH, albumin, glucose, and sediment) before the study. Exclusion criteria were any abnormality in TABLE
1.
FUNCTION
109
the medical and/or menstrual history and in the prestudy physical examination. No other active medication, including steroidal contraceptives, were allowed for at least 3 months before the study. The population studied had a mean age of 28.2 yr (range, 18.7-36.8 yr), a mean weight of 62 kg (range, 50-96 kg), and a mean quetelet index of 22 kg/m2 (range, 1731 kg/m*). The meanparity was 0.6 (range, O-3). Study protocol
Twenty-eight women (study A) were followed during 1 prestudy control cycle, 4 medication months, and 2 poststudy control cycles. Cycle evaluations included BBT, LH, FSH, estradiol (EJ, and progesterone(P,) estimates.Prestudy cycle evaluation included BBT. Hormone determinations were performed in the first and fourth medication months, daily in the midcycle period (days 12-16), and every 3 days during the remaining days of the cycle. In the secondand third medication months and the 2 poststudy control cycles,blood wascollected once in the luteal phase (days 20-24) only. Four women were medicatedwith 300 mg MEL during days 5-17 of the cycle, and 8 women with 300 mg MEL daily (days l-30). In addition, MEL wascombinedwith the synthetic progestin NET. Groups of 4 womenwere medicatedwith 0.75 mg NET in combination with 300, 75, or 7.5 mg MEL. Four women were treated with 75 mg MEL combinedwith 0.3 mg NET. MEL/NET medications were administered for 21 days, followed by 7 pill-free days. In a secondprotocol (study B), 4 womenwere treated for 2 months,with 1 pre- and 1 poststudy control cycle. Two women were treated with 300 mg MEL on days 1-21, and 2 women with 300 mg MEL combinedwith 0.15 mg NET on days 1-21 (Table 1). The study setup wassimilar to the protocol of study A; however, blood samplingand hormone measurementswere performed in ail study months. In addition, transvaginal ultrasound examinations were performed during the prestudy control cycle and the 2 medication months. Prestudy physical examination, electrocardiogram, hematology, biochemistry, and urine analysis were repeatedin the last medication month and in the last poststudy control cycle. In addition, 8 regularly cycling women (mean cycle length, 27.8 days; range, 25-31 days) were sampleddaily as controls (18). The subjectscompleted a weekly questionnaire, which included 15 structured questionsrelated to menstrual, physical, and emotional wellbeing. Hormone
measurements
and statistical
analysis
Blood withdrawal for hormone estimates was performed between0700-1200 h. Plasmaand serumsampleswere frozen
Studydesignandmedicationschemes StudyA
Groupno. No.of subjects MEL (md NET bw) Medicationdays Medicationmonths
4 300
2 8 300
5-17
l-30
4
4
1
3 4 300 0.75 1-21 4
StudvB 4 4 0.75 1-21
5 4 7.5 0.75 1-21
6 4 75 0.3 1-21
4
4
4
75
7 2 300 1-21 21 2
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 October 2015. at 14:40 For personal use only. No other uses without permission. . All rights reserved.
a 2 300 0.15 1-21 2
VOORDOUW ET AL.
110
JCE&M.1992 Vol74.Nol
300 mg Melatonin days l-30 Mean LH decrease LH (IWL)
FIG. 1. Mean LH levels in eight women medicated with 300 mg MEL on days l30 in the first and fourth medication months and eight nonmedicated controls.
30
-
20
-
10 -
I
0
I -3-l cycle
Area 300
I
0 days
from
Under
LH peak
Curve E2 (pmolxdays/L
LH (IUxdays/L) I part A (n=S)
I
(x1000)) 1s
part
B (n=4)
1 16
250
p
Vol. 74, No. 1 Printed
in U.S.A.
Melatonin and Melatonin-Progestin Combinations Alter Pituitary-Ovarian Function in Women and Can Inhibit Ovulation BETTIE C. G. VOORDOUW, ROBERT EUSER, RENE E. R. VERDONK, BERT TH. ALBERDA, FRANK H. DE JONG, AAT C. DROGENDIJK, BART C. J. M. FAUSER, AND MICHAEL COHEN Center for Reproductive Medicine and Breast Cancer Prevention and Departments of Gynecology and Obstetrics (B.C.G. V., B. Th.A., A.C.D., B.C. J.M.F.) and Medicine (F.H. J.), Dijkzigt University Hospital, Rotterdam; the Department of Gynecology and Obstetrics, Academic Hospital (R.E.), Utrecht; and AMR Pharm Holland B. V. (R.E.R. V., M.C.), The Hague, The Netherlands
ABSTRACT. Although melatonin (MEL) controls seasonal reproductive cyclicity in some mammalian species, its role in women is controversial. In this study data are presented related to the influence of MEL or MEL-progestin combinations on the pituitary-ovarian axis and ovulation in 32 women. MEL was administered in a dosage of 300 mg to 12 women for 4 months [to 8 women daily (days l-30) and to 4 women on days 5-17 of the cycle]. MEL was also combined with the synthetic progestin norethisterone (NET) in an attempt to evaluate MEL’s effect on a partially suppressed pituitary-ovarian axis. In 16 women, 4 combinations were tested on 4 women each on davs 1-21: dosages of 300 mg MEL/O.75 mg NET, 75 mg MEL/O.75 mg NET, -7.5 ma MEL/O.75 ma NET. and 75 me MEL/O.30 me NET. In addition, 2 womelwere medicated wgh 300 mg MELalone, and 2 were medicated with 300 mg MEL/O.15 mg NET on days l21 for 2 months. During the study, LH, FSH, estradiol (E,), and progesterone (PJ blood levels were determined at regular intervals. After a period of 4 months, daily administration of 300 mg
T
HE EXACT functions and role of the pineal gland and its major secretory product melatonin (MEL) are still controversial (1). The most persistent and convincing evidence, however, ascribes the pineal gland a role in regulating reproductive events (2). In 1899, Huebner first associated human precocious puberty with various tumours in the pineal region (3). Subsequent evidence for a pineal role in the regulation of reproductive events linked light exposure and pinealectomy with increased ovarian function in the rat (4, 5). The identification of MEL (6) and the effects of environmental light on its biosynthesis (7) suggested MEL to be the link between light-dark periods and reproductive events. The
MEL (days l-30) caused significantly decreased mean LH levels compared to those in 8 nonmedicated controls (P < 0.001). Also compared to nonmedicated control data, a significant inhibition of P, in the first and fourth medication months (P < 0.001) was observed. LH and E, inhibition reached sianificance in the fourth medication month (P c 0.005). Also, thetreatments of 300 mg MEL (days 5-17) and 75 mg MEL combined with 0.3 mg NET caused a significant decrease in LH, EP, and P, levels compared to those in the nonmedicated control group in the first and fourth medication months (P < 0.05). The data further suggest an additive or synergistic effect between MEL and NET. The medications did not alter sleep-wake rhythms and were not complicated by any side-effects. The presented data suggest that MEL and MEL/NET combinations inhibit ovarian function in women, and that MEL/NET combinations can emerge as effective oral contraceptives. (J Ctin Endocrirwl Metab 74: 108-117, 1992)
inhibitory effects of exogenous MEL on ovarian function in several rodent species further support the hypothesis of pineal regulation of reproductive events (8, 9). In contrast with the situation in small rodents, MEL stimulates gonadal function in sheep. In sheep, elevated serum MEL levels are associated with decreasing day length in the fall and introduce the fertile season (10). Stimulatory and inhibitory effects of MEL in sheep and rodents only become apparent after a significant lag time. In addition, the response is complicated by a refractoriness of the hypothalamic-pituitary-gonadal axis to MEL, which develops over time (11). In humans, only scant data exist on the role of MEL in the regulation of reproductive functions. Endogenous MEL secretion follows the light-dark circadian rhythm, with peak nighttime serum levels, and high intensity light can suppress MEL synthesis (12). Whether environmental light or changes in serum MEL levels influ-
Received August 30, 1990. Address all correspondence and requests for reprints to: Mrs. A. C. G. Voordouw, M.D., Center for Reproductive Medicine and Breast Cancer Prevention, Parklaan 12, 3016 BB Rotterdam, The Netherlands. 108
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 October 2015. at 14:40 For personal use only. No other uses without permission. . All rights reserved.
MEL, PROGESTIN,
AND
PITUITARY-OVARIAN
ence reproductive functions in man is uncertain, although a seasonal influence on the ovulatory LH surge has been described (13). Moreover, it is of interest that conception of dizygotic twin pregnancies is more common in the summer months at the time of continuous light in Finland (14), and in primary hypothalamic amenorrhea high nighttime values of MEL have been found (15). The observed negative correlation between MEL secretion and sexual maturation is also consistent with the hypothesis that MEL is antigonadal in man (16). As part of an ongoing program for the development of an estrogen-free MEL-containing oral contraceptive, this study describes the effects of exogenous MEL alone or combined with the synthetic progestin norethisterone (NET) on pituitary-ovarian function in women.
Subjects
and Methods
Participants
The clinical trials were designedin accordancewith WHO guidelinesfor phaseI and II clinical trials in the development of steroidal contraceptives (17). Thirty-two women received medication for 2-4 months, and eight nonmedicated controls were evaluated for one cycle. The studieswere approved by the institutional review boards for experimentation on humans.A detailed informed consentwas obtained from the experimental subjects.The entire study population was recruited by advertisements, and subjectswere assignedrandomly to the study groups.The pharmaceutical combinationsused were prepared in compliancewith pharmacopoeiaguidesand were tested for bioavailability in uiuo. Inclusion criteria required an age between 18-37 yr, a history of regular menstrual cycles between 22-34 days, a biphasic basal body temperature (BBT) in a prestudy control cycle, a normal menstrual pattern of 3-7 bleeding days, and good general health, as determined by a normal medical history, physical examination, normal electrocardiogram,normal standard hematology (Hb, Ht, erythrocyte sedimentation rate, mean cellular volume, mean cellular hemoglobin, meancellular hemoglobinconcentration, and differentiation), biochemistry (sodium, potassium, creatinin, urea, bilirubin, alkaline phosphatase,SGOT, SGPT, lactate dehydrogenase,rGT, total protein, triglycerides, high and low density lipoproteins, total cholesterol, glucose,total Tq, and free
T,) and urine evaluation (pH, albumin, glucose, and sediment) before the study. Exclusion criteria were any abnormality in TABLE
1.
FUNCTION
109
the medical and/or menstrual history and in the prestudy physical examination. No other active medication, including steroidal contraceptives, were allowed for at least 3 months before the study. The population studied had a mean age of 28.2 yr (range, 18.7-36.8 yr), a mean weight of 62 kg (range, 50-96 kg), and a mean quetelet index of 22 kg/m2 (range, 1731 kg/m*). The meanparity was 0.6 (range, O-3). Study protocol
Twenty-eight women (study A) were followed during 1 prestudy control cycle, 4 medication months, and 2 poststudy control cycles. Cycle evaluations included BBT, LH, FSH, estradiol (EJ, and progesterone(P,) estimates.Prestudy cycle evaluation included BBT. Hormone determinations were performed in the first and fourth medication months, daily in the midcycle period (days 12-16), and every 3 days during the remaining days of the cycle. In the secondand third medication months and the 2 poststudy control cycles,blood wascollected once in the luteal phase (days 20-24) only. Four women were medicatedwith 300 mg MEL during days 5-17 of the cycle, and 8 women with 300 mg MEL daily (days l-30). In addition, MEL wascombinedwith the synthetic progestin NET. Groups of 4 womenwere medicatedwith 0.75 mg NET in combination with 300, 75, or 7.5 mg MEL. Four women were treated with 75 mg MEL combinedwith 0.3 mg NET. MEL/NET medications were administered for 21 days, followed by 7 pill-free days. In a secondprotocol (study B), 4 womenwere treated for 2 months,with 1 pre- and 1 poststudy control cycle. Two women were treated with 300 mg MEL on days 1-21, and 2 women with 300 mg MEL combinedwith 0.15 mg NET on days 1-21 (Table 1). The study setup wassimilar to the protocol of study A; however, blood samplingand hormone measurementswere performed in ail study months. In addition, transvaginal ultrasound examinations were performed during the prestudy control cycle and the 2 medication months. Prestudy physical examination, electrocardiogram, hematology, biochemistry, and urine analysis were repeatedin the last medication month and in the last poststudy control cycle. In addition, 8 regularly cycling women (mean cycle length, 27.8 days; range, 25-31 days) were sampleddaily as controls (18). The subjectscompleted a weekly questionnaire, which included 15 structured questionsrelated to menstrual, physical, and emotional wellbeing. Hormone
measurements
and statistical
analysis
Blood withdrawal for hormone estimates was performed between0700-1200 h. Plasmaand serumsampleswere frozen
Studydesignandmedicationschemes StudyA
Groupno. No.of subjects MEL (md NET bw) Medicationdays Medicationmonths
4 300
2 8 300
5-17
l-30
4
4
1
3 4 300 0.75 1-21 4
StudvB 4 4 0.75 1-21
5 4 7.5 0.75 1-21
6 4 75 0.3 1-21
4
4
4
75
7 2 300 1-21 21 2
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 October 2015. at 14:40 For personal use only. No other uses without permission. . All rights reserved.
a 2 300 0.15 1-21 2
VOORDOUW ET AL.
110
JCE&M.1992 Vol74.Nol
300 mg Melatonin days l-30 Mean LH decrease LH (IWL)
FIG. 1. Mean LH levels in eight women medicated with 300 mg MEL on days l30 in the first and fourth medication months and eight nonmedicated controls.
30
-
20
-
10 -
I
0
I -3-l cycle
Area 300
I
0 days
from
Under
LH peak
Curve E2 (pmolxdays/L
LH (IUxdays/L) I part A (n=S)
I
(x1000)) 1s
part
B (n=4)
1 16
250
p
