1 Endocrine Institute, Haemek Medical Center, Afula 18101, 2 Sleep Research Center, 3 Endocrine Laboratory, Rambam Medical Center, Haifa and 4 The B.Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel
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Abstract |
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Key words: follicle-stimulating hormone/ß inhibin/luteinizing hormone/melatonin/testosterone
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Introduction |
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Although the impact of melatonin in human reproduction is still questionable, the current body of information suggests that melatonin and the reproductive system are inter-related (Brzezinski et al., 1988; Luboshitzky et al., 1996
; Kadva et al., 1998
).
The effect of melatonin administration on the pituitary-gonadal hormones secretion in adult men has been studied during the last 25 years and revealed no inhibitory effects on reproductive function (Nordlund and Lerner, 1977; Weinberg et al., 1980
; Strassman et al., 1987
; Webb and Puig-Domingo 1995
; Brzezinski, 1997
). The previously reported effects of melatonin often involved higher doses, or treatment in combination with steroids and did not examine the pulsatile secretion of the pituitary-gonadal hormones (Voordouw et al., 1992
). The effect of melatonin on reproductive hormones may include changes in gonadotrophin-releasing hormone (GnRH) secretion at the arcuate nucleus level and the subsequent pulsatile secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Sizonenko and Aubert, 1986
). Based on studies in women in whom exogenous melatonin enhanced LH concentrations during the follicular phase of the menstrual cycle, it was suggested that by modifying
-aminobutyric acid (GABA), serotonin, dopamine, opioid peptides or prostaglandin activities involved in GnRH regulation, melatonin may enhance GnRH secretion (Cagnacci et al., 1995
).
In several studies examining the effects of melatonin on sleep, the importance of the time of administration in the later afternoon hours has emerged (Garfinkel et al., 1995; Reid et al., 1996
). In children, a dose of 2.510 mg per day given for several years successfully alleviated sleep disorders. However, possible side-effects on growth and development were not studied in these children (Jan and O'Donnell, 1996
).
It is postulated that similar to its hypnotic effects, melatonin may also affect reproductive function when given at a precise circadian phase. The aim of the present study was to test whether doses of melatonin taken to alleviate symptoms of jetlag or shift work affect reproductive function in normal adult men.
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Materials and methods |
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Study protocol
The study comprised 3 experimental nights, 1 month apart. Readings from the first night period served as a no treatment baseline after which subjects were given placebo or 6 mg melatonin orally, once a day at 1700 h for 1 month in a double-blind cross-over placebo controlled fashion. During the treatment periods, subjects maintained regular sleeping habits (23000700 h). Subjects then spent 2 further study nights in the sleep laboratory. On each experimental night, an i.v. catheter was inserted in an antecubital vein kept patent by a slow infusion of 0.9% NaCl. Blood samples (2 ml) were collected every 15 min from 1900 to 0700 h. Subjects were awake between 1900 and 2200 h and remained in bed with lights on (~150 Lux at eye levels). From 2200 to 0700 h, lights were off. Conventional sleep recordings were obtained to verify sleep quality.
Medications
To ensure that the commercial preparation used in this study (Melatone, Cardiovascular Research Ltd, Concord, CA, USA) provided pharmacological serum concentrations, a different group of five adult males (aged 23.8 ± 1.9 years) were given 6 mg melatonin, orally, at 1700 h. Serum melatonin concentrations were determined every 30 min from 1900 to 2300 h. Results were compared to placebo in these subjects.
Analysis of sleep stages
Electrodes were attached for the following electrophysiological recordings: two electroencephalograms (EEG leads C3A2, C4A1), two electro-oculograms and one electromyogram of the mentales. Sleep stages were scored in 30-s epochs according to conventional criteria (Rechtschaffen and Kales, 1968). The following parameters were determined: total recording time (TRT), sleep latency (time from lights off until 3 consecutive minutes of stage 2), actual sleep time (AST = TRT sleep latency + making periods), rapid eye movement (REM) latency or time to first REM (time from beginning of sleep to the first REM episode), percentages of stage 2, stages 3/4 and stage REM and sleep efficiency index (AST/TRT).
Hormone measurements
Blood was centrifuged for 10 min at 2000 g, immediately separated and stored at 20°C until assayed. Serum LH and FSH concentrations were determined by immunoradiometric technique (Biodata Diagnostics, Rome, Italy). The LH intra-assay coefficients of variation (CV) were 4.1% and 3.2% for low (2.23.3 IU/l) and high (2741 IU/l) concentrations respectively. The inter-assay CVs were 3.7 and 0.8% respectively. The sensitivity of the assay was 0.15 IU/l. The FSH intra-assay CVs were 2.7 and 3.8% for low (2.64.0 IU/l) and high (2545 IU/l) concentrations respectively. The interassay CVs were 3.9 and 2.0% respectively. The sensitivity of the assay was 0.5 IU/l. Serum testosterone concentrations were determined by radioimmunoassay (Diagnostic Products Corp, Los Angeles, CA, USA). The intra-assay CVs were 6.0 and 3.0% for low (2.24.0 nmol/l) and high (29.462.0 nmol/l) concentrations respectively. The inter-assay CVs were 1.9 and 1.6% respectively. The sensitivity of the assay was 0.15 nmol/l. Serum inhibin ß concentrations were measured in pooled aliquots of equal volumes of the 49 samples in each experimental night, using a double-antibody enzyme-linked immunosorbent assay (Serotec Ltd, Kidlington, Oxford, UK) with a sensitivity of 15 pg/ml and CV of 46% within plate and 1518% between plates. The reason for measuring inhibin ß on a single sample of the pooled aliquots rather than on every sample is based on previous studies showing that inhibin pulses were undetectable in peripheral blood (Winters, 1990). Serum melatonin concentrations were determined by radioimmunoassay (Bühlmann Lab., Albschwill, Switzerland). The assay sensitivity was 2 pmol/l. The intra-assay CVs were 4.9 and 5.8% for low (412 pmol/l), and high (42106 pmol/l) concentrations respectively. The inter-assay CVs were 7.8 and 6.7% respectively.
Statistical analysis
Six subjects were randomly assigned to a two-period crossover experiment to test the effect of monthly melatonin treatment on serum LH, FSH, testosterone and inhibin ß concentrations. Prior to the experiment, baseline hormone concentrations were determined. The integrated nocturnal LH, FSH and testosterone values were determined as the area under the curve (AUC) from 1900 to 0700 h.
Pearson correlation coefficients between all pairs of the hormone data were compared for each treatment condition. The computer program ULTRA (Van Cauter, 1988) was used to determine the number, duration and mean absolute increment of pulses for LH, FSH and testosterone using a 2CV threshold. Differences between placebo and melatonin treatments were analysed for treatment and carry-over effects, as well as time trend, by analysis of variance. In addition, paired t-tests were performed to test the differences between baseline and placebo as well as between baseline and melatonin.
Polysomnographic data were analysed by paired t-tests for the differences between baseline and melatonin and between baseline and placebo conditions. Placebo and melatonin treatments were compared by analysis of variance (ANOVA) of a crossover design. The following parameters were determined: total recording time (TRT), sleep latency (SL, time from lights off until 3 consecutive minutes of sleep stage 2), actual sleep time [AST = TRT (SL + waking periods)], percentages of stage 2, stage 3/4 and stage REM and sleep efficiency index (%).
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Results |
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Discussion |
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The time of administration is critical for the activity of melatonin on its receptors especially for long-term melatonin treatment. This window of time sensitivity of melatonin receptors in both rats and humans is between 1700 and 2000 h (Guardiola-LeMaître, 1997). Several studies have shown that after an oral dose of 25 mg melatonin, concentrations reached pharmacological values within 1 h and decreased progressively to basal physiological values within 812 h (Wright et al., 1986
; Cagnacci et al., 1991
; Guardiola-LeMaître, 1997
).
Previous studies have revealed that for clock-related problems, 5 mg per day were more efficient than lower doses of melatonin (Arendt, 1996). However, inter-individual differences to melatonin are reflected in a different degree of effect, from a robust sleepiness in some individuals to a lack of any effect in others, independent of the dose used (Zhdanova and Wurtman, 1997
).
Several studies revealed that melatonin administration had no effect on the reproductive hormones in adult men (Fideleff et al., 1976; Nordlund and Lerner, 1977
; Waldhauser et al., 1987
). Wright et al. (Wright et al., 1986
) studied the effect of 2 mg melatonin given at 1700 h for 34 weeks on the FSH, LH and testosterone secretion in adult men and found no effect. However, in this study, hormone concentrations were determined every 46 h, therefore not allowing evaluation of pulsatile secretion of these hormones. Anderson et al. (Anderson et al., 1993
) also failed to detect any effect of 100 mg oral melatonin given daily at 1600 h for 14 days on LH, FSH and testosterone concentrations. In this study, daily plasma melatonin concentrations (determined at 0900 h) were highly elevated (above 1000 pmol/l). The addition of testosterone propionate to melatonin treatment, however, potentiated testosterone suppression of LH. The authors suggested that long duration melatonin may be inhibitory in men, although it takes weeks to change hypothalamic sensitivity (Anderson et al., 1993
). High daily melatonin doses or inappropriate timing of melatonin by dividing the daily dose may induce high melatonin concentrations over a 24 h period that are above the physiological values (Waldhauser et al., 1987
; Cagnacci et al., 1991
; Anderson et al., 1993
).
Disappearance of the normal circadian rhythm may have led to desensitization of melatonin receptors at the concentration of the suprachiasmatic nucleus and the hypothalamus, thus explaining lack of effect of melatonin on the reproductive hormones in these studies.
In the present study both dose and time of melatonin administration were chosen to maximize the possibility of affecting the neuroendocrine system in as near a physiological manner as possible. Yet, the mean nocturnal concentrations of gonadotrophin-gonadal steroid hormones and their pulsatile secretion were not affected by melatonin. However, it is possible that at higher doses, at different times of the day and for longer periods than 1 month as was done in this study, melatonin may be inhibitory to reproduction in men (Anderson et al., 1993; Arendt, 1997).
In contrast, melatonin treatment given to normal cycling women (Voordouw et al., 1992) and in postmenopausal women (Aleem et al., 1984
) successfully suppressed LH and oestradiol concentrations, even when very high doses were given (75300 mg per day at 2100 h). The suppression of LH and oestradiol occurred only after 4 months of melatonin administration (Voordouw et al., 1992
). Also, when given in the morning, melatonin enhanced LH pulse amplitude without changing LH pulse frequency (Cagnacci et al., 1991
).
In conclusion, it was shown that, timed, low dose long-term melatonin administration had no effect on pituitary-gonadal hormones in men. These observations suggest that the use of melatonin in alleviating symptoms of jetlag and some circadian based sleep disorders (Arendt et al., 1997; Palm et al., 1997
), or for other scientifically unfounded indications such as acquired immunodeficiency syndrome, cancer and anti-ageing (Repport and Weaver, 1995; Arendt, 1996
) has no adverse reproductive effects in men.
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Acknowledgments |
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Notes |
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Reference |
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Submitted on July 27, 1999; accepted on October 4, 1999.