Estrogen therapy during menopause improves climacteric symptoms such as hot flushes and sweating. In addition, the therapy protects against osteoporosis1 and cardiovascular disease2 and might have beneficial effects on the central nervous system (CNS).3 Although there is no conclusive evidence that estrogen therapy improves depressed mood in clinically depressed patients, there is some evidence of minor improvements in well-being in healthy female subjects. For instance, estradiol (E2) treatment has been shown to enhance mood in healthy, naturally postmenopausal women without climacteric symptoms.4 Studies performed by Campbell and White-head5 also suggest an independent beneficial psychologic effect of estrogen in less symptomatic postmenopausal women. Sherwin6 found that mood is positively correlated to circulating E2 levels in generally healthy, surgically menopausal, nondepressed women. Finally, mood might be affected differently by different estrogen doses. When naturally menopausal, healthy women were given different doses of estrogen, a higher energy level and a more enhanced sense of well-being were reported with the higher estrogen dose.7
To protect the endometrium from hyperplasia and malignancies in women with intact uteri, estrogen therapy always is combined with continuous or sequential progestogens. However, progestogens given in combination with E2 have been shown to counteract the effect of estrogen on mood.8 Adverse effects caused by progestogens constitute a major problem in hormone replacement therapy (HRT). Among women who discontinue HRT, 35% do so because of negative mood effects.9 A few studies suggest that the adverse effects might be related to the type of progestogen used. For instance, in a recent study involving postmenopausal women, norethindrone acetate caused more negative mood symptoms than did medroxyprogesterone acetate (MPA).10
The serotonin system is involved in numerous physiologic functions, such as appetite, sleep, sexuality, irritability, and pain. Dysfunction of serotonergic transmission has been regarded as an important mechanism in several neuropsychiatric disorders, particularly depression and anxiety.11,12 Sex steroid treatment during menopause and its relation to serotonergic measures has been studied previously, however, in most cases, the focus of these studies has been on estrogen treatment only. A positive correlation has been reported between total plasma estrogen concentration and free plasma levels of the serotonin precursor tryptophan in postmenopausal women.13 In peri- and postmenopausal women, platelet serotonin content was positively correlated to plasma E2 concentration,14 and E2 treatment increased urinary excretion of the serotonin metabolite 5-hydroxyindoleacetic acid.15,16 This increase was abolished, however, when estrogen was combined with norethindrone acetate. The relationship between sex hormones and the serotonin system also seems obvious in the CNS; an overall decrease in serotonin2A receptor density was found in the brains of ovariectomized rats.17 This decrease could be rectified with E2 treatment.
In humans, uptake of serotonin in platelets and uptake of serotonin in brain transmitter terminals are similar active transport processes.18 Furthermore, the transporter proteins in platelets and in the CNS are encoded by the same gene and have amino acid sequences that are identical.19 In the present study, [3H]paroxetine was chosen to label the platelet serotonin transporter because its affinity and amount of specific binding to the transporter are considerably higher compared with [3H]imipramine, another commonly used ligand.20,21 Alterations in the number of platelet [3H]paroxetine binding sites have been found in conditions such as obsessive-compulsive disorder,22 panic disorder,23 unipolar depression,24 and seasonal affective disorder.25
As for the serotonin transporter, the serotonin2A receptor in platelets is similar to that in the human brain.26 In several psychiatric disorders, including depression, specific changes in platelet serotonin2A characteristics have been reported.27 In some earlier studies of the serotonin2A receptor, [3H]ketanserin was used as radioligand. However, [3H]lysergic acid diethylamide ([3H]LSD) is now considered a more appropriate ligand for the serotonin2A receptor in platelets.28
Given that estrogen treatment is considered to improve mood during HRT, and given the influence of E2 on the serotonin system, the purpose of the current study was to examine whether platelet [3H]paroxetine and [3H]LSD binding were influenced across different hormonal phases of HRT. More specifically, the aims of the present study were to examine whether estrogen treatment has modulatory effects on platelet [3H]paroxetine and [3H]LSD binding, to determine whether any change produced by estrogen with regard to platelet binding of [3H]paroxetine and [3H]LSD would be counteracted by the progestogen addition, and to compare the platelet binding of [3H]paroxetine and [3H]LSD between two commonly used progestogens, norethindrone acetate and MPA.
MATERIALS AND METHODS
Over 9 months, 37 women with climacteric symptoms were screened for inclusion in the study. They were recruited through advertisement in the local newspaper. Of these, 29 healthy women were included. Subjects were more than 6 months postmenopausal, had climacteric symptoms, and had concentrations of FSH in serum greater than 30 IU/L. The women had intact ovaries and had not received any HRT in the 3 months before inclusion in the study. They had no contraindications to HRT and were considered physically healthy. The exclusion criterion was treatment with any steroid compounds, benzodiazepines, or other psychotropic drugs for at least 6 months before enrollment in the study. The presence of psychiatric disorders and drug abuse was evaluated using a structured psychiatric interview, Primary Care Evaluation of Mental Disorders, which has been validated for use in primary care settings and conforms to diagnostic criteria in Diagnostic and Statistical Manual of Mental Disorders, 4th Edition.29 Three women fulfilled criteria for major depressive disorder, but otherwise, subjects did not have ongoing mood or anxiety disorders. Patients with drug abuse were excluded. Before inclusion, physical and gynecologic examinations, including transvaginal ultrasound investigations, were performed, and FSH levels were determined. Patients gave written informed consent. The Research Ethics Committee, University of Umeå, Umeå, Sweden, and the Swedish Medical Products Agency approved the study.
HRT was administered in a randomized, double-blind, placebo-controlled crossover design. The women received four 28-day cycles. Estradiol valerate 2 mg was given continuously throughout the study period. The first treatment cycle was a run-in cycle, to give the women time to familiarize themselves with HRT. During this cycle, 10 mg of MPA was added on days 15–28. In the following three randomized cycles, the last 14 days of E2 treatment was combined with 10 mg of MPA, 1 mg of norethindrone acetate, or placebo. Apoteksbolaget Production & Laboratories (the National Corporation of Swedish Pharmacies) in Umeå prepared the identical-looking capsules. After inclusion, patients were assigned to the three different treatments in a sequence determined by a computerized random-number generator. The order of study medication was randomized in blocks of six. During the study, the subjects and study personnel were not informed about the order of treatments. Randomization codes were kept secret at the pharmacy at Umeå University Hospital until completion of the study. Study drugs were packaged in 28-day blister cards by the pharmacy at Umeå University Hospital.
The primary outcome measures of the study were the [3H]paroxetine and [3H]LSD binding kinetics. Blood samples for platelet [3H]paroxetine as well as [3H]LSD binding were taken at four occasions during the study period: before treatment (pretreatment), during estrogen treatment combined with placebo (estrogen only, E2), during estrogen treatment combined with MPA (E2 + MPA), and during estrogen treatment combined with norethindrone acetate (E2 + norethindrone acetate). Blood samples were obtained before treatment and during the last week of each treatment phase. Compliance was assessed by counting the remaining capsules and measuring the concentrations of sex hormone–binding globulin and FSH in serum at each visit.
Throughout the study, patients performed daily symptom ratings using a modified form of the Cyclicity Diagnoser. The Cyclicity Diagnoser, a scale designed for diagnosing cyclical symptoms, has been validated for diagnosis of premenstrual dysphoric disorder.30 The modified Cyclicity Diagnoser covered four physical symptoms (breast tenderness, hot flushes, abdominal bloating, and withdrawal bleeding) and seven psychologic symptoms (cheerfulness, friendliness, libido, anxiety or tension, irritability, fatigue, and depressed mood). The effects on daily life caused by symptoms were graded. The Cyclicity Diagnoser is a Likert scale, graded from 1 to 9, with 1 indicating complete absence of a particular symptom and 9 representing the maximal severity of the symptom. After completing the study, each patient underwent another physical and gynecologic examination and was given the possibility of continuing HRT. Blood was sampled from the antecubital vein with a 20-gauge needle and collected into polyethylene tubes containing 1.6 mg of ethylenediaminetetra-acetic acid per milliliter of blood for the platelet binding assays. All samples were taken between 7:30 and 10:00 AM. Simultaneously, blood samples were taken for analysis of FSH, sex hormone–binding globulin, and E2 in serum.
The method for platelet [3H]paroxetine binding assay has been presented in detail previously.31 In brief, platelet-rich plasma was obtained by centrifugation at 180g for 15 minutes at 20C, and the same amount of assay buffer (50 mM of Tris hydrochloride, 120 mM of sodium chloride, pH 7.4) was added (1:1). After centrifugation at 3000g for 20 minutes at 4C, the pellet was resuspended in assay buffer, recentrifuged at 3000g for 10 minutes at 4C, and homogenized in 10 + 10 mL of the buffer, using a Kinematica Polytron homogenizer (Lucerne, Switzerland), at setting 6 for 7 seconds. After a final centrifugation at 15,000g for 10 minutes at 4C, the pellets were stored at −70C until assay.
On the day of the experiments, the platelet pellet was resuspended in assay buffer to a final volume of 35 mL. The homogenates were incubated for 60 minutes at 25C in a total volume of 1600 μL, consisting of 750 μL of the tissue homogenate, 750 μL of radioligand, and 100 μL of buffer or drug. Nine concentrations of [3H]paroxetine (15.0 Ci/mmol, DuPont NEN., Boston, MA), ranging from 0.01 to 1.2 nM, were used. Specific binding was defined as the difference between total binding and binding in the presence of 10 μM of citalopram.27 The binding experiments were performed in duplicates. After the addition of 6 mL of ice-cold buffer, the homogenates were filtered rapidly through Whatman GF/C filters (Whatman, Maidstone, UK) using a 24-channel cell harvester (Brandel, Gaithersburg, MD). Finally, the filters were washed with three 6-mL rinses of the buffer. The radioactivity trapped by the filters was determined by liquid scintillation spectroscopy. For the highest paroxetine concentrations, total bound [3H]paroxetine did not exceed 5% of the total radioactivity.
The method used for the [3H]LSD binding assay has been described in detail previously.32 In brief, platelet-rich plasma was obtained by centrifugation at 180g for 15 minutes at 20C. The platelet pellet then was obtained by centrifugation at 1200g for 10 minutes at 10C and was stored frozen at −70C until use. On the day of the experiment, the platelet pellet was resuspended in hypotonic Tris buffer, homogenized, centrifuged at 30,000g for 15 minutes, washed, homogenized, centrifuged once more, and suspended in the incubation buffer. Thereafter, aliquots of the preparation were incubated in triplicate for 4 hours at 37C with seven concentrations of [3H]LSD (76.7 Ci/mmol; DuPont NEN.), ranging from 0.25 to 2.5 nM. Nonspecific binding was assessed in the presence of 300 nM of spiperone. The binding was terminated by cell harvester filtration through Whatman GF/C filters, prewashed in a 0.3% solution of polyethylenimine. The radioactivity trapped by the filters was determined by liquid scintillation spectroscopy. Total bound [3H]LSD did not exceed 2% of the total radioactivity. For both radioligand binding methods, final protein concentrations were measured according to Lowry et al33 with modifications suggested by Markwell et al.34
Follicle-stimulating hormone concentrations in serum were measured using a microparticle enzyme immunoassay on AxSYM (Abbott Laboratories, Chicago, IL). The assay sensitivity was 0.37 IU/L. Total coefficients of variation were 4.2% at 7.95 IU/L and 5.2% at 31.9 IU/L. Sex hormone–binding globulin concentrations in serum were analyzed on Immulite (Diagnostic Products Corp., Los Angeles, CA). The assay sensitivity was 3 nmol/L. Total coefficients of variation were 6.0% at 26 nmol/L and 11.3% at 87.6 nmol/L. Estradiol concentrations in serum were analyzed on Immulite. The assay interval was 73–7300 pmol/L. Total coefficients of variation were 11.1% at 194 pmol/L and 10.8% at 531 pmol/L.
The study had an effect size of 0.36, corresponding to the ability to detect a 15% difference in [3H]paroxetine and [3H]LSD binding between treatments with α = .05 and β= .20, based on a previously reported standard deviation within cells of 20% of the mean binding values.32
Because the first treatment cycle was used as a run-in cycle, only the data from the randomized treatment cycles were included in the analyses. The mean scores of daily symptom ratings during the last week of each treatment were compared using the Wilcoxon rank-sum test. Because the study was not powered to detect any differences in adverse mood effects between MPA and norethindrone acetate, mood scores for E2 + MPA and E2 + norethindrone acetate cycles were combined and the cycles called progestogen cycles.
The number of platelet receptors (Bmax), or the affinity of the radioligand to the receptor (Kd), in the [3H]paroxetine and [3H]LSD binding experiments were determined by least-squares linear regression analysis of Scat-chard plots. Because Kd values for both binding experiments were found to be log normally distributed, these values were log transformed before the analyses. First, period effects and carryover effects were evaluated. Period effects measured whether the serotonin measures differed between pretreatment conditions and the last study month (Wilcoxon rank-sum test). Carryover effects measured the difference in serotonin markers between E2, E2 + MPA, and E2 + norethindrone acetate treatment cycles in chronologic order (Kruskal-Wallis test). For the carryover and period effects, P < .1 was considered significant. One-way analysis of variance with repeated measures was used to evaluate the effect of treatment on serotonin measures and serum E2 concentrations. The independent factor was treatment (pretreatment, E2, E2 + MPA, and E2 + norethindrone acetate). Whenever analysis of variance indicated a significant effect of treatment, post hoc tests (using the Tukey honestly significant difference test) were performed. Two-way analysis of variance with repeated measures was used to evaluate possible differences between groups. In these analyses, treatment was used as a within-subjects factor and group was used as a between-subjects factor (study group compared with depressed patients). Because the number of depressed patients was small, we did not perform any post hoc tests. Occasional missing values were interpolated. SPSS (Statistical Package for Social Sciences; SPSS Inc., Chicago, IL) was used for all analyses. For the treatment effects, P < .05 was considered significant. All values in the text and tables are displayed as mean ± standard error of the mean.
Twenty-nine postmenopausal women were included in the study. Three women subsequently dropped out during the run-in month, because of side effects of HRT. Three patients had major depression; their results were analyzed separately. Hence, the study group consisted of 23 patients. One patient had incomplete daily symptom ratings, but otherwise all patients had complete data. Table 1 presents the demographic and physical characteristics of the study group. Follicle-stimulating hormone levels decreased from 69.9 ± 6.0 IU/L to 30.4 ± 3.1 IU/L (P < .1), and sex hormone–binding globulin levels increased from 51.5 ± 5.3 nmol/L to 74.1 ± 7.4 nmol/L (P < .01). There was also a significant increase in the concentration of circulating E2 during the study (F3,66 = 4.42, P < .01, Table 2). To confirm prior reports of mood enhancing effects by E2 treatment,10 mean scores for a number of positive and negative mood symptoms were compared between treatments. Mean scores for depressed mood, irritability, tension, cheerfulness, and friendliness before treatment and during the last week of each treatment cycle are shown in Figure 1. Compared with pretreatment, the estrogen treatment significantly decreased scores for depressed mood (P < .01), irritability (P < .05), and tension (P < .001). The addition of progestogen ended this improvement, in that it caused an increase in self-ratings for depressed mood (P < .001), irritability (P < .05), and tension (P < .05). Estrogen treatment improved the scores for cheerfulness (P < .001) compared with pretreatment, and this improvement was ended with the addition of progestogen (P < .05). The symptom scores for friendliness showed a decrease during the estrogen-progestogen cycles compared with estrogen alone (P < .01), and also a decrease compared with pretreatment values (P < .05). A majority of the subjects did not score the symptom libido, and data on this symptom therefore were not included in the analysis.
Mean data for Bmax and Kd for [3H]paroxetine binding are presented in Table 2. There were no significant changes in Bmax or Kd for [3H]paroxetine binding between pretreatment conditions, E2, E2 + MPA, or E2 + norethindrone acetate treatment (F3,66 = 0.66 and F3,66 = 1.81, respectively). Mean data for Bmax and Kd for [3H]LSD binding also are presented in Table 2. The different treatment settings did not induce any significant changes in Bmax or Kd for [3H]LSD binding (F3,66 = 0.55 and F3,66 = 1.36, respectively). Because the analysis of variance tested all possible interactions between treatments, the nonsignificant findings for [3H]paroxetine and [3H]LSD binding indicated that there were no differences between E2 and E2 + MPA or between E2 and E2 + norethindrone acetate treatment. Likewise, there were no significant differences between E2 + MPA and E2 + norethindrone acetate treatment in [3H]paroxetine and [3H]LSD binding.
Three women had major depression; their data therefore were analyzed separately. These women's pretreatment values for Bmax and Kd for [3H]paroxetine binding were 528.6 ± 42.5 fmol/mg protein and 0.028 ± 0.009 nM, respectively. The pretreatment values for Bmax and Kd for [3H]LSD binding were 64.4 ± 36.5 fmol/mg protein and 2.45 ± 1.02 nM, respectively. Before HRT, depressed women did not differ in any binding characteristics from the study group. The changes in Bmax and Kd for [3H]paroxetine and [3H]LSD binding during the different treatment periods compared with the pretreatment values are presented in Figure 2. HRT induced a significantly more pronounced decrease in Bmax for [3H]LSD binding in these depressed subjects compared with the study group (F1,24 = 4.68, P < .05). Among the depressed patients, Kd for [3H]LSD binding also decreased during HRT; however, this difference between the two groups did not reach statistical significance (F1,24 = 1.95). [3H]paroxetine binding alterations did not differ between the groups during estrogen or estrogen-progestogen treatment (Bmax: F1,24 = 3.47, P = .075; Kd: F1,24 = 0.037, P = .848) (Figure 2).
The main finding of the present study was that estrogen treatment, with or without the addition of progestogen, did not affect the number of, or affinity to, specific platelet binding sites for [3H]paroxetine or [3H]LSD. Furthermore, there were no differences in binding to the platelet serotonin transporter or serotonin2A receptor between the two commonly used progestogens, MPA and norethindrone acetate. The lack of differences between treatments are most likely not due to a type II error, given that the study had a power of 80% to detect clinically relevant differences between treatments. The lack of any differences also is not due to failure of treatment to induce expected mood changes. Whereas the serotonin measures were not affected by HRT, the daily symptom ratings were. The patients experienced an improvement in mood symptoms during estrogen-only cycles. This improvement disappeared with the addition of progestogen. These findings are in line with previous reports of minor beneficial effects of estrogen treatment on well-being and depressed mood symptoms.4,5 Apparently, even though the estrogen-progestogen treatment induced the expected, small, but nevertheless significant changes in mood, these changes were not reflected by alterations in platelet serotonin measures. Furthermore, even though the changes in mood were small, one scale step on the Cyclicity Diagnoser is usually enough to detect a difference in mood.35
Prior reports on postmenopausal estrogen treatment and its interactions with the serotonin transporter or the serotonin2A receptor are scarce and include inconsistent results. In surgically menopausal women treated with estrogen, an up-regulation of platelet [3H]imipramine binding sites has been reported.36 However, in naturally postmenopausal women treated with an E2 implant, Best et al37 could not detect any significant changes in platelet [3H]imipramine or [125I]LSD binding compared with pretreatment. No previous studies have investigated the contribution of adding progestogen to estrogen therapy on platelet [3H]paroxetine or [3H]LSD binding. However, ovarian steroids, in different settings, have been proved to affect serotonin uptake and serotonin2A receptor binding. [3H]imipramine binding has been shown to decrease in the luteal phase compared with the follicular phase of the menstrual cycle,38,39 and [3H]LSD binding has been reported to be significantly increased in the 2nd week of the menstrual cycle in regularly menstruating women compared with oral contraceptive users.32
The anucleated platelet has a life span of 10–14 days, and its characteristics are determined by events in the megakaryocyte that occurred weeks earlier. Thus, it is possible that changes induced by HRT could have occurred if the blood sampling had been scheduled differently—for instance, during the 1st week of the subsequent treatment cycle. In studies involving patients with premenstrual dysphoric disorder, lower platelet [3H]imipramine binding has been detected during the follicular phase, approximately 1–2 weeks after the peak of premenstrual symptoms in the luteal phase.40 Nevertheless, the length of treatment with estrogen-only cycles should be sufficient to detect any estrogen-induced changes, and consequently, only progestogen-induced changes could have been concealed by the turnover time of platelets. Another reason for the lack of effect of estrogen on the serotonin measures could be that even longer treatment times are required to detect a change in platelet [3H]paroxetine and [3H]LSD binding.
Three patients had major depression and were not included in the original study group. Although no conclusions can be drawn from the data from these three patients, it was obvious that they responded differently from the study group to estrogen-progestogen treatment. During estrogen treatment, as well as during estrogen-progestogen treatment, there was a significantly more pronounced decrease in Bmax for [3H]LSD binding in this group compared with the study group. This decrease mimics the response to maprotiline41 and sertraline42 treatment by depressed patients (a single-point [3H]ketanserin binding tended to decrease throughout the treatment course). There is no conclusive evidence, mainly because of the lack of randomized controlled studies, that estrogen treatment is contributory to the pharmacologic management of major depression. In a 6-week, placebo-controlled, randomized trial, E2 replacement was superior to placebo for the treatment of perimenopausal depression, independent of its salutary effects on vasomotor symptoms.43 In another study involving elderly depressed women, estrogen augmented the clinical response to fluoxetine treatment.44 However, because only the fluoxetine treatment was randomized in the study, and because other investigators have found no additive effect of estrogen treatment with regard to antidepressant therapy,45 these findings should be interpreted with caution. Nevertheless, the preliminary results of decreased serotonin2A receptor binding in depressed postmenopausal patients during HRT necessitate future studies.
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