J. Pineal Res. 2000; 28:111–118

Changes in nocturnal melatonin secretion inperimenopausal women: Correlation withendogenous estrogen concentrations

Okatani Y, Morioka N, Wakatsuki A. Changes in nocturnal melatoninsecretion in perimenopausal women: Correlation with endogenous es-trogen concentrations. J. Pineal Res. 2000; 28:111–118. © Munksgaard,Copenhagen

Abstract: Although age-related decrease in melatonin secretion inhumans and animals is well documented, there is a paucity of data onthe precise changes in melatonin secretion that occur during theperimenopausal period. The present study was designed to measurechanges in nocturnal melatonin and to characterize the role played byestrogen in controlling nocturnal melatonin secretion in perimenopausalwomen. Nocturnal serum melatonin concentrations were determinedevery 2 hr in 46 premenopausal women, 44 postmenopausal women,and 11 premenopausal women with uterine leiomyoma scheduled forhysterectomy and bilateral salpingo-oophorectomy. Nocturnal serummelatonin secretion in premenopausal women declined moderately from17 to 45 years of age, and increased during the period from 46 to 50years of age. Among postmenopausal women, a steep, age-relateddecline in nocturnal melatonin secretion was found for up to 15 yearspostmenopause, followed by an extremely gradual decline thereafter. Asignificant negative correlation was observed between the peak serummelatonin concentration and the serum 17 b-estradiol concentration inpremenopausal women aged 40–50 years (r= −0.661, PB0.0005).Daily oral administration of conjugated estrogen (0.625 mg) topostmenopausal women suppressed nocturnal melatonin secretion(PB0.005). A low estrogen state, induced by oophorectomy ofpremenopausal women with uterine leiomyoma led to an increase innocturnal melatonin secretion (PB0.0001). Our findings suggest thattransient elevated nocturnal melatonin secretion during menopause maybe related to the existence of a low estrogen environment. Theage-related decrease in melatonin secretion observed in other conditionsis most likely attributable to other age-related factors.

Yuji Okatani, Nobuyuki Moriokaand Akihiko WakatsukiDepartment of Obstetrics and Gynecology,Kochi Medical School, Kochi, Japan

Key words: aging – estrogen – melatonin –menopause

Address reprint requests to Yuji Okatani,M.D., Department of Obstetrics and Gyne-cology, Kochi Medical School, Oko,Nankoku, Kochi 783-8505, Japan.

Received March 9, 1999;accepted May 12, 1999.

Introduction

Melatonin is synthesized by the pineal gland, andboth synthesis and secretion follow a circadianrhythm, with low rates of production and releaseduring the day and high rates of production andrelease at night. The adrenergic induction of N-acetyltransferase (NAT) activity at night is respon-sible for this diurnal variation [Reiter, 1991].

An age-related decline in melatonin secretionand a negative correlation between melatonin se-cretion and age from childhood to senescence hasbeen reported[Iguchi et al., 1982; Nair et al., 1986;Sack et al., 1986; Waldhauser et al., 1988; Sharmaet al., 1989; Bojkowski and Arendt, 1990; Vakkuriet al., 1996]. Waldhauser et al. [1988] have re-

ported the existence of an age-dependent biphasicdecrease in nocturnal serum melatonin after in-fancy, consisting of a steep decline from earlychildhood to adolescence and a moderate declinein old age. The authors speculated that the steepdecline in the nocturnal serum melatonin concen-tration during the period from early childhood toadulthood is caused by an increase in the mela-tonin distribution volume without an increase inthe rate of secretion. The subsequent linear declinein the nocturnal serum melatonin concentration inthe elderly was believed to have been attributableto a decreasing activity of the pineal gland, whichis a process associated with aging.

Since the identification of gonadal steroid recep-tors in the rat pineal gland [Luttge and Wallis,

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1973; Cardinali et al., 1974, 1975], evidence hassuggested that melatonin synthesis also may bemodulated by the gonadal steroids [Houssay andBarcelo, 1972; Cardinali et al., 1981a; Arendt etal., 1983]. Previous studies in this laboratory havedemonstrated that the decline in melatonin synthe-sis that occurs during puberty may be, in part,related to an increase in the estrogen concentra-tion [Okatani et al., 1997]. Furthermore, the in-hibitory effect of estrogen on melatonin synthesisappears to be mediated by the modulation ofNAT activity [Okatani et al., 1998a,b] and adeny-late cyclase activity [Okatani et al., 1998c]. Inhumans, the effect of gonadal steroid on pinealsecretion of melatonin remains controversial.Brzezinski et al. [1988] found no change in plasmamelatonin levels during the menstrual cycle. Thenocturnal secretion of melatonin is markedly in-creased in women with secondary amenorrheawhose estrogen levels are extremely low [Berga etal., 1988; Brzezinski et al., 1988; Okatani andSagara, 1994]; exogenous estrogen stimulationsuppressed such melatonin secretion [Okatani andSagara, 1994]. However, Bartsch et al. [1995] havereported that both transdermal and oral estradioltreatment led to an increase as well as decrease ofmelatonin production in different postmenopausalwomen.

Earlier studies on the circadian rhythm of mela-tonin synthesis and secretion in aging focused onpuberty and senescence. However, solid data onthe precise change in melatonin synthesis duringthe perimenopausal period, when ovarian estrogenproduction decreased markedly, are rare. In theprevious study, we found a temporary increase inpineal melatonin synthesis during the peri-menopausal period in rat [Okatani et al., 1999].We also found that the increased melatonin syn-thesis may result from decreasing levels of endoge-nous estrogen during this period.

The present study was, therefore, undertaken toestablish the role of estrogen in the control ofnocturnal melatonin synthesis in perimenopausalwomen. We first determined the change in noctur-nal melatonin secretion in these women. There-after, we studied the influences of oophorectomyand the administration of estrogen on nocturnalmelatonin secretion in these women.

Material and methods

Subjects

The study population consisted of 46 pre-menopausal women (aged 17–50 years) and 44postmenopausal women (aged 48–90 years). The

subjects were volunteers and written informedconsent was obtained from all women prior toenrollment in the study. The subjects were askedto give their menstrual records in order to assessthe reproductive state. The mean age in post-menopausal women was 50 years. The study de-sign was approved by the Ethics Committee ofKochi Medical School. Eleven premenopausal pa-tients with uterine leiomyoma (aged 42–50 years)had undergone total simple hysterectomy and bi-lateral oophorectomy. All women were nonsmok-ers and had no cardiovascular disease or diabetesmellitus. The subjects received no hormonal treat-ment for at least 1 year before the study. Studieson the premenopausal women were carried outbetween the 6th and 8th day of the follicularphase. The data were pooled according to four agegroups in premenopausal women: 17–29 years(group A, n=12), 30–39 years (group B, n=9),40–45 years (group C, n=10), and 46–50 years(group D, n=15). Postmenopausal women weredivided into five groups according to the post-menopause period: 1–5 years (group E, n=8),6–10 years (group F, n=6), 11–15 years (groupG, n=7), 16–20 years (group H, n=9), and20–40 years (group I, n=14).

Experimental design

Study 1. The nocturnal changes in the serum mela-tonin concentration were determined in volun-teers. No food intake was permitted during thetest period. The subjects remained in a sound-at-tenuated room and ate a meal at 18:00 hr. Theywere instructed to lie down beginning at 18:30 hrand were exposed to a intense light of \300 lux ateye level. Lights were turned off at 21:00 hr, andall subjects were asleep by midnight. Lights wereturned on at 06:00 hr, at which time the subjectsawoke and they remained awake thereafter. Bloodsamples (4 mL) were drawn through an indwellingvenous cannula at 2-hr intervals between 20:00and 08:00 hr.

Study 2. The effect of estrogen on nocturnal mela-tonin secretion was studied in seven post-menopausal volunteers who had climactericsymptoms (aged 48–52 years). Conjugated estro-gen (Premarin 0.625 mg/day; Weiss, Tokyo,Japan) was administered orally to each subject for8 wk. The interval between studies 1 and 2 was 2months. Blood samples were obtained via an in-dwelling venous cannula at 2-hr intervals between20:00 and 08:00 hr. The light and sleep/awakeconditions were the same as for study 1.

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Study 3. Eleven premenopausal patients with uter-ine leiomyoma (aged 42–50 years) were studied todetermine the relation between the endogenousserum estrogen concentration and melatonin secre-tion. Total simple hysterectomy and bilateraloophorectomy was performed in each patient un-der general anesthesia. Nocturnal blood samplingwas obtained before and 4 wk after the operation.Blood sampling and the light and sleep/awakeconditions were the same as for study 1.

Assays

Serum was separated within 1 hr of blood sam-pling and stored at −20°C until the assays for 17b-estradiol (E2) and melatonin were performed.The concentrations of serum E2 were measured induplicate using a commercially available radioim-munoassay kit (MIS; Bristol, Tokyo, Japan).Serum E2 was measured in the 20:00 hr samplesonly as there normally are no changes in serum E2

levels over a 24-hr period. Intra- and interassaycoefficients of variations determined on pooledhuman serum were less than 10%. The lower limitof detection for serum E2 was 37 pmol/L. Sampleswith estrogen concentrations below the limit ofdetection were reported as 37 pmol/L. The serummelatonin concentration was determined by highperformance liquid chromatography with electro-chemical detection, as previously described[Sagara et al., 1998]. Melatonin in serum (1 mL)was extracted with 12 volumes of chloroform.After addition of 0.1 M NaOH (100 mL), themixture was shaken for 10 min and centrifuged.Chloroform layer was taken and washed twicewith 2 mL of distilled water. After further cen-trifugation, the chloroform layer was collected anddried under vacuum. The residue was dissolved in0.5 mL of chloroform three times and dried undera stream of nitrogen. The residue was dissolved in25 mL of the eluent and 20 mL was used for theanalysis. Recovery rate of melatonin was 77.793.5% (mean9S.D.). The assay for melatonin wasperformed on a reverse-phase QC pack C-18column (IRICA, Kyoto, Japan), using a mobilephase of 0.1 M phosphate buffer (pH 3.4) contain-ing 21% (v/v) acetonitrile. The assay had a sensi-tivity of 5.0 pg and intra- and interassaycoefficients of variation were less than 10%.

Statistical analysis

Data are expressed as the means9S.E.M. Statisti-cal analyses of results were performed by two-wayanalysis of variance. The significance of intergroupdifferences in the mean melatonin and E2 concen-

trations were analyzed by Student’s t-test orWelch’s t-test. A level of PB0.05 was accepted asstatistically significant.

Results

The serum E2 concentration in groups A, B, C,and D were 148913 pmol/L, 162919 pmol/L,13899 pmol/L, and 9197 pmol/L, respectively.The difference in the E2 concentration betweengroups C and D was significant (P=0.0066). Theserum E2 concentration in postmenopausal womenwas below the lower limit of detection. The noc-turnal profiles of the mean serum melatonin con-centration in premenopausal women andpostmenopausal women are shown in Figs. 1 and2. An age-related decrease in nocturnal melatoninsecretion was found in groups A, B, and C. Mela-tonin secretion in group A was greater than ingroup C (F=3.345, P=0.0034), and melatoninsecretion in group B was greater than in group C(F=2.372, P=0.0034). Also, the maximumserum melatonin concentration was higher ingroup A than in group B (P=0.0033), and higherin group B than in group C (P=0.0271). How-ever, nocturnal melatonin secretion was signifi-cantly greater in group D than in group C(F=2.276, P=0.0394). The maximum serum

Fig. 1. Sequential changes in the nocturnal serum melatoninconcentration in premenopausal women: �, women aged 17–29 years (n=12); �,women aged 30–39 years (n=9); ,women aged 40–45 years (n=10); and , women aged46–50 years (n=15). Conditions were normal, undisturbedsleep in the dark. Lights were off between 21:00 and 06:00 hr.Blood samples were collected at 2-hr intervals between 20:00and 08:00 hr. Data are reported as the means. Bars indicateS.E.M.

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Fig. 2. Sequential changes in the nocturnal serum melatoninconcentration in postmenopausal women. Women were di-vided into five groups according to the interval sincemenopause: , 1–5 years (n=8); �, 6–10 years (n=6); ,11–15 years (n =7); �, 16–20 years (n=9); �, 21–40 years(n=14). Conditions and blood sample collection were asdescribed in Fig. 1. Data are reported as the means. Barsindicate S.E.M.

The mean preoperative serum E2 concentrationin patients who underwent oophorectomy was12497 pmol/L. Serum E2 concentration followingoophorectomy was below the detection limit of theassay. The patterns of melatonin secretion prior toand following oophorectomy were different (F=8.747, PB0.0001, Fig. 5). The maximum mela-tonin concentrations were higher postoperativelythan preoperatively (PB0.0001).

Discussion

Our findings show that the nocturnal serum mela-tonin concentration increases transiently duringmenopause. This increase in nocturnal melatoninsecretion during the menopausal period seems tobe closely related to ovarian function and is influ-

Fig. 3. Correlation between peak nocturnal serum melatoninand serum E2 concentrations in premenopausal women aged17–39 years (upper) and 40–50 years (lower). Data are re-ported as the means. Bars indicate S.E.M.

melatonin concentration was significantly higherin group D than in group C (PB0.0001). Amongpostmenopausal women, a steep, age-related de-cline in nocturnal melatonin secretion was foundfor up to 15 years postmenopause, followed by anextremely gradual decline thereafter. The patternof nocturnal melatonin secretion was different ingroups E and F (F=2.461, P=0.0309), in groupsF and G (F=2.883, P=0.0138), and in groups Gand I (F=3.214, P=0.0058). The maximumserum melatonin concentration was also differentin groups E and F (P=0.0010), in groups F andG (P=0.0014), and in groups G and I (P=0.0002). The maximum serum melatonin concen-tration consistently occurred between 02:00 and04:00 hr in all groups. A significant correlationwas noted between the serum E2 concentrationand the peak serum melatonin concentration inpremenopausal women aged 40–50 years (r= −0.661, P=0.0003; y=0.72894−2.9534x), but notamong women less than 40 years old (Fig. 3).

The mean serum E2 concentrations in patientswho received Premarin for 8 wk was 125913pmol/L. The pattern of melatonin secretion wasdifferent prior to and during Premarin administra-tion (F=3.543, P=0.0036, Fig. 4). Maximummelatonin concentration during Premarin admin-istration was significantly lower than the concen-tration prior to treatment (PB0.0001).

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Fig. 4. Sequential change in the nocturnal serum melatoninconcentrations in postmenopausal women (n=7) prior to(), and during (�) treatment with conjugated estrogen.Conjugated estrogen 0.625 mg/day was administered orally for8 wk. Conditions and blood sample collection were as de-scribed in Fig. 1. Data are reported as the means. Bars indicateS.E.M.

to postmenopausal women suppressed the eleva-tion in the melatonin concentration. The presentresults in humans were consistent with those in theprevious animal study [Okatani et al., 1999]. Thehigher melatonin concentrations in the postopera-tive period may not be related to operation-in-duced factors. A noradrenaline uptake inhibitorreportedly increases melatonin production in hu-mans [Skene et al., 1994]. However, since thepineal gland receives primarily by postganglionicsympathetic innervation, the pineal appears not toparticipate in the stress response. In addition, at 4wk after operation, patients have a normal lifeactivity and an undisturbed sleep/wake cycle. Anyclear phase shift in the diurnal melatonin rhythmwas not evident.

The effects of estrogen on pineal function variesmarkedly, depending on the species, dose of estro-gen, and duration of estrogen administration.Consequently, no consensus has been reached con-cerning the effect of estrogen on melatonin secre-tion. However, some investigators have proposeda negative feedback loop between estrogen andmelatonin during the estrous cycle in the rat[Ozaki et al., 1978; Johnson et al., 1982; Alonso etal., 1993]. Arendt et al. [1983] found that ovariec-tomy was followed by an increase in melatoninsecretion in the ewe. In humans, nocturnal mela-tonin secretion increased markedly in women withsecondary amenorrhea [Berga et al., 1988; Brzezin-ski et al., 1988]. We have also found that noctur-nal melatonin secretion in women with secondaryamenorrhea is enhanced as a function of the sever-ity of hypothalamic-pituitary-gonadal dysfunction[Okatani and Sagara, 1994]. Similarly, estrogendeficiency, resulting from gonadotropin-releasinghormone agonist therapy in patients with en-dometriosis who have normal menstrual cycles,causes an elevation in the nocturnal melatoninconcentration [Okatani and Sagara, 1994]. Otherinvestigators, however, found a lack of effect ofestrogen on melatonin secretion in women[Brzezinski et al., 1988; Bartsch et al., 1995].

The effect of estrogen on pineal activity may beexerted on the presynaptic noradrenergic input orat a subsequent step in the pathway of melatoninsynthesis. A previous study in this laboratory indi-cated that neither estrogen administration norovariectomy changes pineal norepinephrine con-centration in female rats [Okatani et al., 1997].Also, pineal norepinephrine concentration in fe-male rats does not change between 4 and 24months of age [Okatani et al., 1999]. These find-ings suggest that estrogen effects do not involvethe modulation of noradrenergic input. Previousdata, however, indicate that the effects of estrogen

enced primarily by the endogenous estrogen envi-ronment. This hypothesis is supported by the find-ings that oophorectomy in premenopausal womenresulted in an increase in nocturnal melatoninsecretion, and that the administration of estrogen

Fig. 5. Sequential changes in the nocturnal serum melatoninconcentration in premenopausal women with uterine leiomy-oma (n=11) prior to (), and 4 wk following (�) hysterec-tomy and oophorectomy. Conditions and blood samplecollection were as described in Fig. 1. Data are reported as themeans. Bars indicate S.E.M.

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on melatonin synthesis in peripubertal female ratsis mediated, in part, by the modulation of NATactivity [Okatani et al., 1998a,b], and by nore-pinephrine-induced stimulation of pineal adeny-late cyclase activity [Okatani et al., 1998c].Furthermore, a previous study indicates that atemporal increase in pineal melatonin synthesisduring perimenopausal period in the rat appearsto involve modulation of NAT activity [Okatani etal., 1999]. The hypothesis that estrogen influencesthe norepinephrine-induced stimulation of pinealadenylate cyclase activity is supported by the find-ing of Moujir et al. [1990], who demonstrated thattreatment with 17 b-estradiol blocks the iso-proterenol-induced increase in rat pineal mela-tonin concentrations. The suppressive effect ofestrogen on the level of pineal adenylate cyclaseactivity also has been demonstrated in female rats[Weiss and Crayton, 1970; Davis, 1978]. In addi-tion, estrogen has been shown to alter the numberof pineal b1 receptors [Weiland and Wise, 1989],which are required for NAT stimulation. Otherpossible mechanism by which estrogen may affectpineal function may involve the pineal membranepotential, DNA-dependent RNA polymerase ac-tivity, or the synthesis of a specific estrogen-in-duced protein, which has been demonstrated inanimals [Cardinali et al., 1974]. Thus, additionalstudy is needed to characterized the effect of estro-gen on pineal function.

Another mechanism underlying the increasedmelatonin synthesis during perimenopausal periodmight include the alteration of serum go-nadotropin levels. In the present study, we did notdetermine the relationship between serum mela-tonin levels and gonadotropin. Fernandez et al.[1990] have reported a negative correlation be-tween day-time serum melatonin levels and folliclestimulating hormone (FSH) during themenopausal period. Cardinali et al. [1987] havereported that FSH decreases the rat pineal mela-tonin content, and that this effect may be exertedmainly through the superior cervical ganglion[Cardinali et al., 1981b]. Pang et al. [1987], how-ever, have reported a positive correlation ofplasma melatonin with FSH in a study of preg-nant women. Additional studies are needed toclarify the effect of FSH on melatonin synthesisduring perimenopausal period. As for the clear-ance and excretion of melatonin, no significantdecrease was found during the menopausal period[Fernandez et al., 1988].

The decline in the nocturnal serum melatoninconcentration that occurs during the interval from17 to 45 years of age, and the decline that occurspostmenopause, may be related to pineal aging,

because no significant changes in the serum estro-gen concentration take place at this time. Skene etal. [1990] and Vakkuri et al. [1996] have alsoobserved two steps in the decline of urinary mela-tonin excretion during perimenopause: the firstone when moving from premenopausal women(B40 years) to women of 40–44 years, and thesecond one when moving from women of 50–54years to women of 55–59 years. These observa-tions are consistent with the present findings. Incontrast to our results, they did not find anychanges in urinary melatonin excretion between 45and 54 years. The discrepancy between the presentfindings and the reports of Skene et al. [1990] andVakkuri et al. [1996] is difficult to reconcile. Themechanisms involved in the age-associated reduc-tion in pineal function are unknown. However,Greenberg and Weiss [1978] have demonstratedthat the density of the b-adrenergic receptors inthe rat pinealocyte membrane declines with age.Henden et al. [1992] also demonstrated that al-though the b-adrenergic receptor affinity for theradioligand was not altered with age, the densityof receptors in the pineal gland of 28-month-oldmale rats was only 50% that of the pineal of youngadult males. An age-associated biphasic decline innocturnal melatonin concentrations post-menopause may be the results of multiple factors.

It is not known whether the transient increase innocturnal melatonin secretion that occurs duringmenopause has any physiologic significance or ismerely a physiologic consequence of changes inthe gonadal–pineal feedback system. Recently,several investigators hypothesized that melatoninplays a role in aging and age-related disease [Re-iter et al., 1994]. Studies in rats [Dilman et al.,1979] and mice [Pierpaoli and Regelson, 1994;Reiter, 1995] have suggested that decreased mela-tonin secretion may be associated with an acceler-ation of the aging process. Melatonin may provideprotection against aging by attenuating the effectsof cell damage by free radicals [Reiter, 1995] or viaimmunoenhancement [Maestroni, 1993]. Mela-tonin is a powerful scavenger of oxygen free radi-cals, quenching hydroxyl radicals [Tan et al., 1993;Matuszek et al., 1997], peroxyl radicals [Pieri etal., 1994], and highly toxic peroxynitrite anion[Cuzzocrea et al., 1997]. Free radical generationmay increase as a consequence of normal aging, oralternatively the defense system evolved to combatoxidative stress, including antioxidative enzymes,may diminish in effectiveness [Floyd and Carney,1993; Reiter, 1995]. In addition to its direct an-tioxidant effects, a recent report has demonstratedthat melatonin increases the mRNA levels forglutathione peroxidase, copper zinc superoxide

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dismutase, and manganese superoxide dismutasein the rat cerebral cortex [Kotler et al., 1998]. Aprevious study in this laboratory demonstratedthat hydrogen peroxide impaired the activity ofovarian aromatase, which is the key enzyme ofestrogen biosynthesis, and that a rapid decrease inovarian glutathione peroxidase and superoxidedismutase activity in humans takes place duringmenopause [Okatani et al., 1993]. Melatonin mighthave a supplemental role in estrogen synthesis inhuman ovary as an antioxidant. Furthermore, wefound that administration of a physiologic dose ofmelatonin to female senescence-accelerated miceprevents the age-related oxidative DNA damage inthe brain [Morioka, et al. 1999]. However, thistopic requires additional studies to establish thepathophysiologic role of melatonin during peri-menopause and the climacteric.

Acknowledgments

This work was supported by Research Grant 70145142from the Ministry of Education of Japan.

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