Effects of Sleep Fragmentation and Estradiol Decline on Cortisol in a Human Experimental Model of Menopause

Aviva Y Cohn; Leilah K Grant; Margo D Nathan; Aleta Wiley; Mathena Abramson; Jessica A Harder; Sybil Crawford; Elizabeth B Klerman; Frank A J L Scheer; Ursula B Kaiser; Shadab A Rahman; Hadine Joffe

doi:10.1210/clinem/dgad285

. 2023 May 19;108(11):e1347–e1357. doi: 10.1210/clinem/dgad285

Effects of Sleep Fragmentation and Estradiol Decline on Cortisol in a Human Experimental Model of Menopause

Aviva Y Cohn ^1,^2,³, Leilah K Grant ^4,^5,⁶, Margo D Nathan ⁷, Aleta Wiley ^8,⁹, Mathena Abramson ^10,¹¹, Jessica A Harder ¹², Sybil Crawford ¹³, Elizabeth B Klerman ^14,^15,^16,¹⁷, Frank A J L Scheer ^18,¹⁹, Ursula B Kaiser ²⁰, Shadab A Rahman ^21,^22,^#, Hadine Joffe ^23,^24,^#,^✉

¹ Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital (BWH), Harvard Medical School (HMS), Boston, MA 02115, USA

² Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

³ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

⁴ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

⁵ Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA

⁶ Division of Sleep Medicine, HMS, Boston, MA 02115, USA

⁷ Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

⁸ Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

⁹ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

¹⁰ Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

¹¹ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

¹² Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

¹³ Tan Chingfen Graduate School of Nursing at UMass Chan Medical School, Worcester, MA 01605, USA

¹⁴ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

¹⁵ Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA

¹⁶ Division of Sleep Medicine, HMS, Boston, MA 02115, USA

¹⁷ Department of Neurology, Massachusetts General Hospital, HMS, Boston, MA 02114, USA

¹⁸ Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA

¹⁹ Division of Sleep Medicine, HMS, Boston, MA 02115, USA

²⁰ Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital (BWH), Harvard Medical School (HMS), Boston, MA 02115, USA

²¹ Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA

²² Division of Sleep Medicine, HMS, Boston, MA 02115, USA

²³ Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA

²⁴ Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA

Shadab A Rahman and Hadine Joffe contributed equally to this work.

^✉

Correspondence: Hadine Joffe, MD, MSc, Women’s Hormones and Aging Research Program, Department of Psychiatry, Brigham and Women’s Hospital, 75 Francis St, Thorn 11-11, Boston, MA 02115, USA. E-mail: hjoffe@bwh.harvard.edu.

PMCID: PMC10584010 PMID: 37207451

Abstract

Context

Perturbations to the hypothalamic-pituitary-adrenal (HPA) axis have been hypothesized to increase postmenopausal cardiometabolic risk. Although sleep disturbance, a known risk factor for cardiometabolic disease, is prevalent during the menopause transition, it is unknown whether menopause-related sleep disturbance and estradiol decline disturb the HPA axis.

Objective

We examined the effect of experimental fragmentation of sleep and suppression of estradiol as a model of menopause on cortisol levels in healthy young women.

Methods

Twenty-two women completed a 5-night inpatient study during the mid-to-late follicular phase (estrogenized). A subset (n = 14) repeated the protocol after gonadotropin-releasing hormone agonist-induced estradiol suppression. Each inpatient study included 2 unfragmented sleep nights followed by 3 experimental sleep fragmentation nights. This study took place with premenopausal women at an academic medical center. Interventions included sleep fragmentation and pharmacological hypoestrogenism, and main outcome measures were serum bedtime cortisol levels and cortisol awakening response (CAR).

Results

Bedtime cortisol increased 27% (P = .03) and CAR decreased 57% (P = .01) following sleep fragmentation compared to unfragmented sleep. Polysomnographic-derived wake after sleep-onset (WASO) was positively associated with bedtime cortisol levels (P = .047) and negatively associated with CAR (P < .01). Bedtime cortisol levels were 22% lower in the hypoestrogenized state compared to the estrogenized state (P = .02), while CAR was similar in both estradiol conditions (P = .38).

Conclusion

Estradiol suppression and modifiable menopause-related sleep fragmentation both independently perturb HPA axis activity. Sleep fragmentation, commonly seen in menopausal women, may disrupt the HPA axis, which in turn may lead to adverse health effects as women age.

Keywords: menopause, cortisol, sleep fragmentation, estradiol, stress, hypothalamic-pituitary adrenal axis

Menopause marks a pivotal health transition in female aging. This transition is characterized by estradiol decline, and is frequently associated with vasomotor symptoms and sleep disturbance (1, 2). Cardiometabolic risk is increased in postmenopausal women, for which there are many proposed contributory mechanisms (3), including perturbations to the hypothalamic-pituitary-adrenal (HPA) axis (4). Disruption of cortisol dynamics manifesting as alterations in the daily rise and fall of cortisol can lead to adverse health effects (5). Two key cortisol measures are disrupted in common health conditions: (i) higher than expected cortisol at bedtime is seen in pathologic conditions such as insomnia and depression, and is associated with increased risk of impaired glucose tolerance and cardiovascular mortality (6), and (ii) blunting of the morning rise in cortisol, known as the cortisol awakening response (CAR), is evident in some disease states, including hypertension, cardiovascular disease, chronic pain, and sleep disorders, compared to healthy controls (7, 8). Sleep and estradiol independently modulate circulating cortisol levels (9, 10). Therefore, perturbation of cortisol secretion as a consequence of menopause-related sleep disturbance, which manifests as middle-of-the-night interruption called sleep fragmentation, and estradiol decline may be an important mediator of increased cardiometabolic risk in aging women.

Sleep disruption is a common complaint in women during the menopausal transition. Up to 60% of perimenopausal women have sleep difficulties (11). During the perimenopausal transition, women typically report frequent nighttime awakenings—that is, increased wake after sleep onset (WASO) without necessarily a reduction in total sleep time (TST) or increased difficulty falling asleep at the beginning of the night (12). This middle-of-the-night fragmentation of sleep during the menopausal transition is explained primarily by nighttime vasomotor symptoms (VMS) (13). Whether sleep fragmentation during the menopausal transition without concomitant reduction in TST affects HPA axis activity is unknown. Some studies in patients with untreated obstructive sleep apnea or chronic insomnia in which middle-of-the-night awakenings are frequent suggest that 24-hour serum cortisol levels are increased overall when sleep fragmentation occurs (9, 14). Additionally, restriction of sleep to a 3-hour TST in young healthy women increased afternoon and evening cortisol levels and decreased the morning cortisol level 30 minutes after awakening (15). However, these results are confounded by the concomitant exposure to hypoxia among people with sleep apnea, the reduced TST in the people with insomnia, and experimental sleep restriction, and therefore do not directly address the effect of isolated menopausal sleep fragmentation on the HPA axis.

Estradiol decline related to menopause may itself affect cortisol levels. Older postmenopausal women have higher cortisol levels than younger premenopausal women (16); however, the relative contributions of chronological age and of estradiol decline to changes in cortisol levels across menopause are unknown. Evidence from animal models supports estradiol action increasing corticotropin-releasing hormone secretion, thereby inducing adrenocorticotropin and corticosterone secretion both basally and in response to stress (17, 18). However, human studies have shown inconsistent results (19): effects of estrogen replacement therapy on cortisol in postmenopausal women are associated with an increase (10, 20), decrease (21), or no change in basal cortisol levels. Consequently, the effect of estradiol decline on the HPA axis across menopause remains unclear.

Our aim in the present study was to systematically evaluate the independent effects of sleep fragmentation and estradiol decline and their interactions on the HPA axis, with bedtime cortisol levels and CAR as the primary end points, in healthy premenopausal women. To achieve this aim, we experimentally fragmented sleep in premenopausal women to mimic the sleep fragmentation seen in women with the most severe hot flashes, both before and after suppressing estradiol pharmacologically using a gonadotropin-releasing hormone (GnRH) agonist. This experimental paradigm allows for causal inferences of the effects of sleep fragmentation and estradiol suppression on HPA axis function, as well as analysis of within-person changes without the confounding influence of age. Building on data from studies from other sleep conditions and the association between estradiol decline and adverse cardiovascular health (22), we hypothesized that estradiol suppression and sleep fragmentation would each independently perturb these two HPA axis measures so that the bedtime cortisol levels would be elevated and CAR would be blunted.

Materials and Methods

Participants

Twenty-two healthy premenopausal women, aged 18 to 45 years, were studied. During screening procedures, they completed a medical history and physical examination to exclude pregnancy, and cardiometabolic, neurologic, psychiatric, sleep, and HPA axis disorders. Individuals taking systemic hormonal contraception, corticosteroids, centrally active medications or supplements that might affect VMS, sleep, or the HPA axis were excluded. All participants had regular menstrual cycles (range, 25-35 days), confirmed by tracking for at least 2 cycles before the inpatient study. The study was approved by the Mass General Brigham Human Research Committee, and written informed consent was obtained.

Study Design Overview

The study included 2 inpatient visits, each for 6 consecutive days and 5 nights, to the Intensive Physiological Monitoring Unit of the Center for Clinical Investigation at Brigham and Women's Hospital. The first inpatient visit was conducted during the mid- to late-follicular phase of the menstrual cycle as estimated based on cycle length, referred to henceforth as the estrogenized state. During the visit, the first 2 nights consisted of unfragmented sleep followed by 3 nights of experimental sleep fragmentation with frequent awakenings (details described later). A subset of 14 participants then received a GnRH agonist with a one-time open-label intramuscular injection of depot leuprolide 3.75 mg (Lupron Depot, AbbVie Inc) during the mid-luteal phase of the menstrual cycle; this produces a temporary hypoestrogenized state, starting ~2 weeks after the injection and lasting for ~12 weeks. Approximately 4 to 5 weeks post leuprolide injection, participants were readmitted for the second inpatient visit, identical to the first, which will be referred to as the hypoestrogenized state.

Sleep Fragmentation Protocol

During both inpatient visits, participants had an 8-hour sleep opportunity with a fixed sleep/wake schedule (scheduled time in bed 23:00-07:00) for the initial 2 nights of unfragmented sleep. On the subsequent 3 nights, sleep was fragmented during an extended sleep opportunity to preserve TST (1 hour of extra scheduled time in bed 23:00-8:00). On each sleep fragmentation night, participants were scheduled to be awakened 34 times for 2 minutes at a time, to achieve a total of 68 minutes of scheduled WASO, modeling the WASO observed in menopausal women with frequent nighttime hot flash awakenings (23, 24). The sleep fragmentation protocol began 30 minutes after scheduled bedtime, after which participants were awakened every 15 minutes via auditory tones lasting up to 30 seconds, with a sound intensity range of 60 to 90 dB. (See Grant et al. (25) for details of the acoustic stimulus procedure used to fragment sleep.) Once awakened, participants had to maintain wakefulness for a 2-minute duration within each 15-minute interval. Wakefulness was verified by an event marker pressed each time the participant heard the auditory stimulus that was presented every 10 seconds during the 2-minute interval. The last fragmentation interval in the morning ended 15 minutes before scheduled wake to minimize interference with spontaneous wake. At scheduled wake, a laboratory technician woke up the participant.

Objective Sleep Recording

During all inpatient nights, sleep was recorded objectively by polysomnography (PSG) collected using a digital recorder (Vitaport-3 digital recorder, TEMEC Technologies B.V.). Recordings included electroencephalogram, electrooculogram, chin electromyogram, and a 2-lead electrocardiogram. Electrodes were positioned according to the International 10-20 system, with linked mastoid references (Fz, C3, Cz,C4, Pz, Oz). Electrode impedances (<10 kΩ) were checked (OhmMate impedance meter, TEMEC Instruments B.V.) at the beginning and end of the sleep recordings. PSG recordings were visually scored according to the American Academy of Sleep Medicine Scoring Manual (version 2.6) (26) to define the time spent in each sleep stage, including light sleep (stage N1 and N2), deep sleep (stage N3), and rapid eye movement (REM) by a registered polysomnographic technologist.

Hormone Assays

Blood samples were collected throughout the inpatient visits, during both unfragmented and fragmented sleep, via an indwelling intravenous catheter attached to long tubing enabling blood to be drawn from a distance without disrupting participants.

Cortisol

HPA axis metrics included bedtime cortisol levels from samples measured immediately before lights out, and CAR, calculated as the difference in cortisol levels between scheduled wake time and 30 minutes after scheduled wake. For these end points, serum cortisol was assayed in samples collected at bedtime (23:00), scheduled wake time (07:00 on unfragmented nights, 08:00 on fragmented nights), and 30 minutes after scheduled wake time. To assess the effect of sleep fragmentation on bedtime cortisol and CAR, values were compared before and after sleep fragmentation (Fig. 1). For bedtime cortisol, prefragmentation levels were assessed on night 2 and postfragmentation values were assessed on night 5 (ie, after 2 consecutive nights of fragmentation, the final bedtime cortisol available). Prefragmentation CAR values were calculated the morning of day 2, and postfragmentation values were calculated the morning of day 6 (ie, after 3 consecutive nights of fragmentation, the final CAR value available). These data points were selected to maximize the exposure to each sleep condition.

Figure 1. — Study schema. The study protocol consisted of two 5-night inpatient visits: one during the mid-to-late follicular phase, when estradiol levels are relatively high, and a second visit approximately 4 weeks after administration of a gonadotropin-releasing hormone (GnRH) agonist (leuprolide) to induce pharmacologic estradiol suppression as noted in A. Each inpatient visit included 2 nights of unfragmented sleep (solid black bars, UNF) followed by 3 nights of experimental sleep fragmentation (hatched bars, FRA). Bedtime cortisol levels (square) and the cortisol awakening response (CAR) (circle) were evaluated after the second night of the unfragmented condition and the third night of the fragmented condition in both estradiol states depicted in B.

Serum cortisol was assayed by Brigham Research Assay Core (Brigham and Women's Hospital,) via a chemiluminescent immunoassay on a Beckman Access 2 (catalog No. 33600, RRID:AB_2802133). Intra-assay and interassay variations were 4.4% to 6.7% and 6.4% to 7.9% respectively, with a range of detection of 0.4 to 60.0 μg/dL.

Estradiol

Fasting estradiol on day 2 and day 6 was measured in the estrogenized state (visit 1) and in the hypoestrogenized state after leuprolide (visit 2). Serum estradiol was assayed using liquid chromatography–mass spectrometry (Brigham Research Assay Core, Brigham and Women's Hospital). Intra-assay and interassay variation were less than 5% and less than 10% respectively, with a range of detection of 1 to 500 pg/mL.

Data analysis

This is a secondary analysis conducted on data obtained from an ongoing larger study, therefore an a priori sample size was not completed. Data were analyzed using generalized linear mixed models (GLMMs) with sleep fragmentation and estradiol suppression as the fixed effects, and their interaction, and subject-level random effects to accommodate within-subject correlation over time. GLMM with log-normal and normal distribution were used for bedtime cortisol levels and CAR, respectively. In secondary analyses quantitating the magnitude of the effect of specific dimensions of sleep disruption on HPA axis activity, associations between sleep-related outcomes as time-varying covariates (eg, minutes of WASO, minutes of TST) and corresponding HPA axis outcomes (ie, bedtime cortisol levels and CAR) were tested using GLMM. To estimate the main effects of sleep fragmentation and estradiol suppression and their interaction, all available data from 22 participants, 14 of whom were studied in both estradiol states, were analyzed with a single fully adjusted omnibus statistical model. Therefore, the effect estimate for sleep fragmentation is derived from data from 22 participants, whereas the estimates for estradiol suppression and the interaction effect are derived from data from 14 participants. Results are presented as mean ± SD for demographic, baseline characteristics, and sleep architecture, and as least squares (LS) mean ± SE for GLMM results adjusted for sleep condition, estradiol state, and their interaction.

Results

Twenty-two participants completed the inpatient study during their predicted mid-to-late follicular phase (estrogenized state). A subset of 14 participants repeated the study following leuprolide administration (hypoestrogenized state). The remaining 8 did not complete study procedures during the hypoestrogenized state before receiving leuprolide because of changes in personal schedule (n = 4), loss to follow up (n = 2), and changed mind about participating (n = 2). There were no statistically significant differences (all P ≥ .1; t tests for continuous data and χ² for categorical data) in key baseline characteristics (age, race, body mass index, estradiol levels) between the 14 who completed the study and the 8 who dropped out before receiving leuprolide. Data from all available participants were included in the final analysis. Participant demographics stratified by study visit are presented in Table 1.

Table 1.

Baseline characteristics

Participants	Estrogenized state (n = 22)	Hypoestrogenized (n = 14)
Age (mean ± SD), y	31.0 ± 6.5 (21-45)	29.4 ± 5.6 (21-42)
Race (%, N)
White	54.5% (n = 12)	64.3% (n = 9)
Black	13.6% (n = 3)	21.4% (n = 3)
Asian	13.6% (n = 3)	7.1% (n = 1)
Other/not reported	18.2% (n = 4)	7.1% (n = 1)
BMI (mean ± SD)	25.3 ± 3.9 (18.5-31.2)	25.0 ± 4.2 (18.5-31.2)
Estradiol (mean ± SD), pg/mL	88.6 ± 62.0 (18.0-214.0)	7.8 ± 7.1^a (1.2-18.9)
No. (%, N) reporting vasomotor symptoms	N/A	86% (n = 12)

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Abbreviations: BMI, body mass index; N/A, not applicable.

Signifies P less than .05 when compared to the estrogenized state.

Effect of Sleep Fragmentation Protocol on Sleep Architecture

The experimental sleep fragmentation protocol had the intended robust effect on WASO and sleep architecture (Table 2). Experimental sleep fragmentation significantly increased WASO in the estrogenized state compared to unfragmented sleep (103.2 ± 32.9 minutes vs 37.8 ± 26.1 minutes; P < .001). Similarly, the same pattern was found in the hypoestrogenized state (WASO for fragmented sleep 91.4 ± 36.0 minutes vs unfragmented sleep 34.7 ± 17.2 minutes; P < .0001). As expected, there was no change in TST between the unfragmented and fragmented sleep conditions within either estradiol state (estrogenized: unfragmented 425.5 ± 33.2 minutes vs fragmented 420.68 ± 34.21 minutes, hypoestrogenized: unfragmented 426.1 ± 25.1 minute vs fragmented 429.1 ± 40.6 minutes; both P ≥ .47). Additional changes in sleep architecture induced by experimental sleep fragmentation included more N1 sleep, less N3 sleep (both, P < .01) in the estrogenized and the hypoestrogenized state. There was less REM sleep only in the estrogenized state (P = .01) but not in the hypoestrogenized state (see Table 2).

Table 2.

Sleep continuity and sleep architecture in the unfragmented and fragmented sleep conditions

	Estrogenized (n = 22) Mean ± SD		Hypoestrogenized (n = 14) Mean ± SD
	UNF	FRA	UNF	FRA
TST, min	425.45 ± 33.24	420.68 ± 34.21	426.14 ± 25.07	429.05 ± 40.56
WASO, min	37.82 ± 26.1	103.23 ± 32.92^a	34.66 ± 17.19	91.41 ± 36.01^c
N1, min	50.24 ± 15.57	74.33 ± 18.05^a	48.93 ± 19.14	74.9 ± 22.15^c
N2, min	211.52 ± 39.51	222.95 ± 27.37	203.88 ± 22.36	214.74 ± 30.88
N3, min	69.67 ± 33.2	40.02 ± 23.31^a	77.98 ± 30.16	54.32 ± 25.14^c
REM, min	94.02 ± 17.99	83.37 ± 19.22^b	95.36 ± 18.43	85.1 ± 15.07

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Abbreviations: FRA, fragmented sleep; N1, stage 1 sleep; N2, stage 2 sleep; N3, stage 3 sleep; REM, rapid eye movement; TST, total sleep time; UNF, unfragmented sleep; WASO, wake after sleep onset.

Statistical significance of P < .0001 for the estrogenized state

Statistical significance of P < .05 for the estrogenized state

Statistical significance of P < .0001 for the hypoestrogenized state on a Wilcoxon rank sum test.

Effect of Leuprolide on Estradiol Levels

Mean (±SD) estradiol levels peaked at 88.6 ± 62.0 pg/mL in the estrogenized state, and as expected, were significantly reduced to 7.8 ± 7.1 pg/mL (P < .0001) following leuprolide administration.

Effect of Sleep Fragmentation on Bedtime Cortisol and Cortisol Awakening Response

Bedtime cortisol levels were significantly higher (adjusted raw group means increased by 26.9%) in the fragmented sleep condition (adjusted mean ± SE 3.30 ± 0.36 μg/dL) compared to the unfragmented sleep condition (2.60 ± 0.19 μg/dL) for the combined estradiol states (omnibus analysis collapsed across both the estrogenized and hypoestrogenized states); P = .03 (Fig. 2A). The interaction between sleep fragmentation and estradiol status on bedtime cortisol levels was not statistically significant (P = .66). When stratified by estradiol state, there was a significant group difference in bedtime cortisol levels in the hypoestrogenized state between the fragmented and unfragmented sleep conditions (2.85 ± 0.26 μg/dL vs 2.35 ± 0.21 μg/dL, respectively; P = .02). This finding was in the same direction in the estrogenized state but not statistically significant where variability was much greater (bedtime cortisol levels in the fragmented sleep condition 3.83 ± 0.62 μg/dL vs unfragmented 2.89 ± 0.30 μg/dL; P = .13) (Fig. 2B and 2C).

Figure 2. — Effect of sleep fragmentation on bedtime serum cortisol levels. Bedtime serum cortisol levels before sleep fragmentation (UNF), and after 2 nights of sleep fragmentation (FRA) in (A) both estradiol states combined; (B) subset during the estrogenized state; and (C) subset during the hypoestrogenized state. Bar graphs show the least squares (LS) mean ± SEM.

For the combined estradiol states, the adjusted raw group mean for CAR decreased by 56.9% after sleep fragmentation (1.59 ± 0.71 μg/dL) compared to unfragmented sleep (3.69 ± 0.51 μg/dL); P = .01 (Fig. 3A). This change resulted from a significant decline in cortisol levels at the wake + 30 minutes time point (unfragmented sleep: 16.35 μg/dL vs fragmented sleep: 14.29 μg/dL; P < .01), rather than a rise in cortisol at scheduled wake time (unfragmented: 12.63 μg/dL vs fragmented: 12.60 μg/dL; P = .98). There was no statistically significant interaction between sleep fragmentation and estradiol status on CAR (P = .41). When stratified by estradiol state, the CAR decreased significantly after sleep fragmentation in the estrogenized state (fragmented sleep: 0.80 ± 1.26 vs unfragmented sleep: 3.68 ± 0.69 μg/dL; P = .03; Fig. 3B); however, this was not evident in the hypoestrogenized state (fragmented sleep: 2.38 ± 0.89 vs unfragmented sleep: 3.70 ± 0.59 μg/dL; P = .26; Fig. 3C). Sensitivity analyses to assess the main effect of sleep fragmentation on bedtime cortisol and CAR conducted on data from the 14 participants who completed both the estrogenized and hypoestrogenized conditions showed consistent results for sleep fragmentation on bedtime cortisol (P = .07) and CAR (P = .02) compared to results from the data from all 22 women.

Figure 3. — Effect of sleep fragmentation on CAR. CAR before sleep fragmentation (UNF), and after 3 nights of sleep fragmentation (FRA) in (A) both estradiol states combined; (B) subset during the estrogenized state, and (C) subset during the hypoestrogenized state. CAR, change in serum cortisol at scheduled wake + 30 minutes – cortisol level at scheduled wake. Bar graphs show the least squares (LS) mean ± SEM.

Association of Polysomnography-derived Wake After Sleep Onset and Total Sleep Time With Bedtime Cortisol Levels and Cortisol Awakening Response

While WASO was experimentally targeted to reach 68 minutes in the sleep fragmentation condition and TST was intended to be maintained at 8 hours, there was some interindividual variability in the extent to which these targets were achieved; there was similarly some natural interindividual variability in WASO and TST during the unfragmented condition. PSG-derived amount of WASO was significantly positively associated with bedtime cortisol levels (Fig. 4A), with a 1.23 μg/dL higher bedtime cortisol level for each additional hour of WASO both for estrogenized and hypoestrogenized states combined (P = .047). Conversely, there was a negative association between WASO and CAR (Fig. 4B), with a 2.10 μg/dL lower CAR for each additional hour of WASO (P < .01). Neither bedtime cortisol levels (P = .92) nor CAR (P = .10) were associated with PSG-derived TST.

Figure 4. — Relationships of duration of polysomnography-derived wake time after sleep onset (WASO) with bedtime cortisol levels and CAR. Regression plots show (A) bedtime serum cortisol levels relative to polysomnography-derived WASO; and (B) CAR as a function of WASO. CAR, change in cortisol at scheduled wake + 30 minutes – cortisol level at scheduled wake.

Among 72 sleep episodes analyzed (ie, unfragmented and fragmented combined), data were available from 69 (96%) episodes, of which only 17 episodes (∼ 25%) included spontaneous wake before scheduled wake. Furthermore, when specifically examining unfragmented sleep episodes, this frequency was similar where 6 out of 30 (20%) unfragmented sleep episodes had spontaneous wake before scheduled wake. CAR was significantly lower when wake occurred spontaneously prior to scheduled time (−0.38 ± 1.29 vs 3.82 ± 0.37; P = .01); however, sensitivity analyses of CAR adjusting for whether spontaneous wake occurred before scheduled time showed the effects of sleep fragmentation on CAR still remained statistically significant (P = .044) and the correlation between CAR and WASO also remained significant (P = .004).

Effect of low Estradiol State on Bedtime Cortisol Levels and Cortisol Awakening Response

The adjusted raw group mean in bedtime cortisol levels were 22% lower in the hypoestrogenized (2.59 ± 0.21 μg/dL) than in the estrogenized (3.32 ± 0.33 μg/dL; P = .02; Fig. 5A) state. However, CAR did not differ between the hypoestrogenized (3.04 ± 0.49 μg/dL) and estrogenized (2.24 ± 0.8 μg/dL) states (P = .38; Fig. 6A). When stratified by sleep condition, there was a trend toward a lower bedtime cortisol level in the hypoestrogenized compared to the estrogenized state during both unfragmented (2.35 ± 0.21 μg/dL vs 2.89 ± 0.30; P = .11) and fragmented sleep (2.85 ± 0.26 μg/dL vs 3.83 ± 0.62; P = .06), respectively (Fig. 5B and 5C). There were no differences in the CAR between the hypoestrogenized and estrogenized states in either sleep condition (both P ≥ .35; Fig. 6B and 6C). There was no interaction between sleep fragmentation and estradiol status on bedtime cortisol levels or CAR (both P ≥ .41). Additionally, there was no correlation of estradiol levels with either bedtime cortisol levels (P = .25) or CAR (P = .21). Sensitivity analyses to assess the main effect of estradiol suppression on bedtime cortisol and CAR conducted on data from the 14 participants who completed both the estrogenized and hypoestrogenized conditions showed consistent results for estradiol suppression on bedtime cortisol (P = .01) and CAR (P = .69) compared to results from the data from all 22 women.

Figure 5. — Effect of estradiol suppression on bedtime serum cortisol levels. Bedtime serum cortisol levels in the estrogenized state (HiE2), and in the hypoestrogenized state (LoE2) for (A) both sleep states combined; (B) subset during the unfragmented sleep condition (UNF), and (C) subset during the fragmented sleep condition (FRA). Bar graphs show the least squares (LS) mean ± SEM.

Figure 6. — Effect of estradiol suppression on CAR. CAR in the estrogenized state (HiE2), and in the hypoestrogenized state (LoE2) for (A) both sleep states combined; (B) subset during the unfragmented sleep condition (UNF); and (C) subset during the fragmented sleep condition (FRA). CAR, change in cortisol at scheduled wake + 30 minutes – cortisol level at scheduled wake. Bar graphs show the least squares (LS) mean ± SEM.

Discussion

In this study, we examined the independent effects of sleep fragmentation, estradiol suppression, and their interaction on the HPA axis using a pharmacologic model of menopause that reduced estradiol levels to the postmenopausal range, paired with an experimental sleep fragmentation paradigm mimicking the sleep interruption typically observed across the menopause transition. We found that sleep fragmentation increased bedtime cortisol levels (see Fig. 2) and decreased CAR (see Fig. 3), changes in cortisol dynamics known to be associated with poorer health including cardiovascular disease and impaired glucose tolerance (6, 7). Additionally, we found that estradiol suppression decreased bedtime cortisol levels (see Fig. 5) and did not change CAR (see Fig. 6), contrary to the hypothesized adverse effects of menopausal estradiol decline on the HPA axis (22, 27). These results suggest that factors other than estradiol decline itself contribute to adverse cardiovascular health associated with changes in cortisol during menopause. Overall, these changes to cortisol apply clinically: Bedtime cortisol levels are used as a clinical marker of cortisol excess (5), whereas morning cortisol is used as a clinical marker of cortisol sufficiency (28). Alterations to the expected diurnal pattern has been shown to lead to pathological conditions: Elevated bedtime cortisol is associated with increased cardiovascular mortality (29) and diabetes (6), whereas blunted CAR is associated with increased autoimmune, cardiovascular, and psychiatric disease (7). Taken together, these findings provide robust experimental evidence that menopause-related changes in sleep, but not the estradiol decline itself seen with menopause, disrupt cortisol dynamics, potentially providing a modifiable pathway through which menopause may translate to adversely affect cardiometabolic health.

Nighttime VMS, a common complaint during the menopausal transition, correlate with both subjective sleep disturbance (24) and changes in objective sleep measures including the number of awakenings and duration of wakefulness (WASO) on PSG (23). In this innovative study, we aimed to replicate sleep fragmentation experimentally modeling what can occur with the most frequent nighttime VMS (23) to evaluate the effect on HPA axis perturbations as measured by bedtime cortisol levels and CAR. To our knowledge, menopause-pattern sleep disruption due to VMS has not been examined in relation to cortisol dynamics. Previous studies assessing the association between disruption of the HPA axis and menopause symptoms have reported mixed results: Severe VMS were associated with perturbation of cortisol dynamics in some studies (30-32), whereas other studies found no association (33, 34). These conflicting results may be due to nonseparation of day from night VMS and the nature of the observational designs employed in those studies. The strength of our study is the robust method of controlled experimental interventions that allows for disentanglement of the independent effects of sleep fragmentation and estradiol decline across each condition.

Circulating cortisol levels are under endogenous circadian regulation and are directly affected by sleep patterns (35). Prior studies have shown that slow-wave sleep decreases cortisol levels, while brief arousals during sleep lead to increased cortisol secretory bursts at the time of the awakening (9). We found disruption of normal sleep via sleep fragmentation also led to alterations to cortisol levels at time points unrelated to wake episodes themselves. Changes in WASO were associated with both an increase in bedtime cortisol levels and blunting of CAR in the absence of significant reduction in TST (see Fig. 4). These results indicate that this typical pattern of sleep fragmentation (high WASO without shortening of TST) observed in women during the menopausal transition (36, 37) may perturb cortisol dynamics independent of a reduction in TST. Although sleep fragmentation (increased WASO) and sleep deprivation (shortened TST) represent different patterns of sleep disturbance, consistent with our present findings, elevated evening cortisol levels have been reported in prior studies in which sleep was restricted to 4 hours (38, 39). This suggests that the adverse physiologic consequences of elevated bedtime cortisol may be precipitated by either sleep deprivation or sleep fragmentation or their combination. In our study, the magnitude of bedtime cortisol elevation resulting from sleep fragmentation was modest (∼ 0.7 μg/dL, 26.9% increase). Other studies involving sleep restriction have shown elevations in evening cortisol of a similar magnitude (38). While these changes are modest, they are expected to occur repeatedly given the chronic nature of sleep disturbances, and persistent exposure to even small changes to cortisol levels can lead to worse health outcomes (9).

In contrast to bedtime cortisol, the effects of sleep disturbance on CAR is less well characterized: While one study showed reduced CAR with sleep restriction (40), other studies have found no relationship of sleep restriction (41) or sleep fragmentation to CAR (42). Some of the inconsistency in the literature regarding CAR is related to varying methods of cortisol sampling and measurement, as well as confounding influences on CAR including circadian input and the limbic system (7). Overall, we observed that sleep fragmentation induced changes in both bedtime cortisol and CAR, indicating the disruptive effect of sleep fragmentation on HPA axis regulation. Whether the adverse cardiometabolic health effects of sleep disruption (9) are mediated by changes in bedtime cortisol levels and/or CAR, and their specific roles in pathophysiologic pathways, requires further study.

Contrary to the hypothesis that estradiol decline has a detrimental effect on cortisol dynamics, we found that without sleep fragmentation, GnRH agonist–induced estradiol suppression decreased bedtime cortisol levels with no changes in CAR. Exogenous estradiol administration (to isolate the effect of estradiol on the HPA axis) increases cortisol levels both basally and in response to stress in animal models and humans (17, 20, 36, 43). This stimulatory effect of estradiol on the HPA axis might explain why in our study estrogenized women had higher bedtime cortisol levels compared to the postleuprolide hypoestrogenic state. While the higher bedtime cortisol with sleep fragmentation seen when our study participants were estrogenized might counterintuitively suggest higher risk for cardiometabolic disease in younger estrogenized women (6, 29), there might be differences in acute estradiol suppression as modeled with leuprolide vs the more prolonged progression to hypoestrogenism observed across the natural menopause transition. We did not find a change in CAR following estradiol suppression, consistent with a previous study that showed no differences in CAR when comparing premenopausal and postmenopausal women with similarly contrasting estradiol levels, together suggesting that CAR may not be directly influenced by estradiol (44).

In our study, when examining the effect of experimental sleep fragmentation stratified by estradiol state, sleep fragmentation significantly increased bedtime cortisol in the hypoestrogenized state, whereas in the estrogenized state, the finding was not statistically significant but was in the same direction. Moreover, we found that CAR was blunted in the estrogenized state, but not in the hypoestrogenized state. While we did not find an interaction effect, these results suggest that the effect of sleep fragmentation on HPA axis activity may be modulated by the concurrent estradiol state; additional studies with adequate statistical power to detect an interaction effect are needed. Taken together, our study findings suggest that experimental sleep fragmentation appears to have a more robust and consistently disruptive effect on HPA axis end points than did estradiol suppression, although we had a smaller sample for our analyses examining estradiol effects. Perhaps some of the inconsistencies examining the HPA axis in the menopausal literature may be attributable to the opposing effects of concurrent menopause-related factors that we were able to disentangle between sleep fragmentation and estradiol suppression.

Strengths of the study include the experimental design allowing for repeated-measures within-subject analysis, which is a robust approach and minimizes the effect of interindividual variability. Additionally, in this mechanistic study, we were able to investigate induced changes in the HPA axis in response to uniform interventions by experimentally controlling sleep and estradiol conditions; this design provides insight that cannot be characterized in cross-sectional studies. Limitations in the study included a smaller sample size for the estradiol state contrasts, as some participants withdrew before leuprolide administration, reducing the hypoestrogenized subgroup. Additionally, our experimental design intentionally maintained TST, limiting the ability to detect TST associations with cortisol dynamics between the unfragmented and fragmented sleep states. By preserving TST and experimentally adding WASO, there was an expected 1-hour delay in scheduled wake time from 7 Am to 8 Am on mornings following sleep fragmentation that may affect CAR findings, as some studies suggest that an earlier wake time can lead to a higher CAR (45); however, CAR has been shown to have high intraindividual stability (41), and other studies have shown no effect of wake time on CAR (45). Additionally, while the possibility for acclimatization exists across and between each inpatient visit, it is difficult to disentangle the effect of acclimatization on bedtime cortisol and CAR between each visit from concomitant changes in estradiol levels, though the fact that each inpatient visit was approximately 4 to 5 weeks apart makes acclimatization less likely. Likewise, while there is a possibility that acclimatization might affect bedtime cortisol and CAR across visit days within each inpatient visit, we chose the cortisol assay time points of maximal difference between the unfragmented and fragmented sleep states to maximize the cumulative effect of sleep fragmentation. Finally, serum total cortisol was assessed, and while cortisol-binding globulin (CBG) is regulated by estradiol, studies show this is specific to exogenous administration of oral estradiol and with very high endogenous estradiol such as in pregnancy, in which other mechanisms of action including the first-pass effect of the liver and pregnancy-specific CBG likely also affect total cortisol levels (46, 47). In a study comparing CBG levels between premenopausal women and postmenopausal women, there were no significant between-group differences in CBG (48), and in a study looking at premenopausal women given leuprolide compared to those who received leuprolide + estradiol add-back therapy, there were likewise no significant changes in CBG levels (49), thus in the range of estradiol in our study, we would not expect CBG levels to vary significantly to affect interpretation of free cortisol levels.

Conclusion

Our results show that sleep fragmentation, seen commonly during the menopause transition, adversely affects HPA axis activity by elevating bedtime cortisol and blunting CAR. Notably, the extent to which both bedtime cortisol and CAR change after sleep fragmentation relates to the amount of increase in objectively assessed WASO. In contrast, estradiol suppression did not increase bedtime cortisol or alter CAR, suggesting that estradiol decline is likely not the key factor influencing HPA axis dysregulation across menopause. Instead, our findings highlight the potential importance of menopause-related sleep fragmentation in disrupting the HPA axis, which in turn may lead to adverse cardiometabolic effects in aging women. This has clinical ramifications, as sleep is often assessed by number of hours slept per night; however, our work indicates that TST is a not the only sleep parameter that should be considered. Understanding the effect of frequent awakenings on cortisol dynamics may provide important insight into the physiologic function of the HPA axis and its effect on health in women, in addition to providing an important therapeutic target for behavioral or pharmacological interventions to treat sleep disturbances that are common across the menopause transition.

Acknowledgments

We thank the study participants as well as the technical, laboratory staff, nurses, and physicians at the BWH Center for Clinical Investigation.

Abbreviations

CAR: cortisol awakening response
CBG: cortisol-binding globulin
GLMM: generalized linear mixed model
GnRH: gonadotropin-releasing hormone
HPA: hypothalamic-pituitary-adrenal
N1: stage 1 sleep
N2: stage 2 sleep
N3: stage 3 sleep
PSG: polysomnography
REM: rapid eye movement
TST: total sleep time
VMS: vasomotor symptoms
WASO: wake after sleep onset

Contributor Information

Aviva Y Cohn, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital (BWH), Harvard Medical School (HMS), Boston, MA 02115, USA; Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA; Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA.

Leilah K Grant, Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA; Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA; Division of Sleep Medicine, HMS, Boston, MA 02115, USA.

Margo D Nathan, Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA.

Aleta Wiley, Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA; Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA.

Mathena Abramson, Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA; Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA.

Jessica A Harder, Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA.

Sybil Crawford, Tan Chingfen Graduate School of Nursing at UMass Chan Medical School, Worcester, MA 01605, USA.

Elizabeth B Klerman, Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA; Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA; Division of Sleep Medicine, HMS, Boston, MA 02115, USA; Department of Neurology, Massachusetts General Hospital, HMS, Boston, MA 02114, USA.

Frank A J L Scheer, Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA; Division of Sleep Medicine, HMS, Boston, MA 02115, USA.

Ursula B Kaiser, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital (BWH), Harvard Medical School (HMS), Boston, MA 02115, USA.

Shadab A Rahman, Division of Sleep and Circadian Disorders, Departments of Medicine and Neurology, BWH, Boston, MA 02115, USA; Division of Sleep Medicine, HMS, Boston, MA 02115, USA.

Hadine Joffe, Women's Hormones and Aging Research Program, Department of Psychiatry, BWH, HMS, Boston, MA 02115, USA; Connors Center for Women's Health and Gender Biology, BWH, HMS, Boston, MA 02115, USA.

Funding

This work was supported by the National Institute of Aging R01AG053838 (principal investigator: H.J., U.B.K., F.A.J.L.S., E.B.K.), NIH-T32DK007529 (U.B.K.), Brigham Research Institute microgrant (A.Y.C.), and Clinical Translational Science Award UL1RR025758 to Harvard University and Brigham and Women’s Hospital from the National Center for Research Resources. The authors were also supported in part by U54AG062322 (H.J., U.B.K., E.B.K.), R01HL140574 (F.A.J.L.S.), K24HL105664 and R01-HD102624 (E.B.K.), and R01HL159207 (S.A.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

Disclosures

A.Y.C., L.K.G., M.D.N., A.W., M.A., S.C., F.A.J.L.S., U.B.K. have nothing to disclose. E.B.K. is a consultant with Circadian Therapeutics, The National Sleep Foundation, Sanofi-Genzyme, and Yale University Press. Her spouse owns Chronsulting. S.A.R. holds patents for (1) Prevention of Circadian Rhythm Disruption by Using Optical Filters, and (2) Improving sleep performance in subject exposed to light at night; S.A.R. owns equity in Melcort Inc, and has received grant/research support from Seoul Semiconductor Co Ltd, Merck & Co, Inc, Pfizer Inc, Vanda Pharmaceuticals Inc, NIH, and NASA. These interests were reviewed and managed by Brigham and Women's Hospital and Mass General Brigham in accordance with their conflict of interest policies. H.J. is a consultant to Bayer, Eisai, Merck, and Hello Therapeutics, and has received grant/research support from NIH, Merck, and Pfizer. Her spouse is an employee at Arsenal Biosciences and has equity in Merck Research Lab. J.A.H. is a consultant to Atman Health, has done medical writing for Centerfield Media Holding Company, and provided expert witness services to O’Rourke & Hawk, LLP.

Data Availability

Some or all data sets generated during and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Some or all data sets generated during and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request.

[dgad285-B1] 1. Kravitz HM, Kazlauskaite R, Joffe H. Sleep, health, and metabolism in midlife women and menopause. Obstet Gynecol Clin North Am. 2018;45(4):679‐694. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dgad285-B2] 2. Woods NF, Mitchell ES. Sleep symptoms during the menopausal transition and early postmenopause: observations from the Seattle Midlife Women's Health Study. Sleep. 2010;33(4):539‐549. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dgad285-B3] 3. Roa-Díaz ZM, Raguindin PF, Bano A, Laine JE, Muka T, Glisic M. Menopause and cardiometabolic diseases: what we (don’t) know and why it matters. Maturitas. 2021;152:48‐56. [DOI] [PubMed] [Google Scholar]

[dgad285-B4] 4. Mezzullo M, Gambineri A, Di Dalmazi G, et al. Steroid reference intervals in women: influence of menopause, age and metabolism. Eur J Endocrinol. 2021;184(3):399‐411. [DOI] [PubMed] [Google Scholar]

[dgad285-B5] 5. Lightman SL, Birnie MT, Conway-Campbell BL. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr Rev. 2020;41(3):bnaa002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dgad285-B6] 6. Hackett RA, Steptoe A, Kumari M. Association of diurnal patterns in salivary cortisol with type 2 diabetes in the Whitehall II Study. J Clin Endocrinol Metab. 2014;99(12):4625‐4631. [DOI] [PMC free article] [PubMed] [Google Scholar]

[dgad285-B7] 7. Fries E, Dettenborn L, Kirschbaum C. The cortisol awakening response (CAR): facts and future directions. Int J Psychophysiol. 2009;72(1):67‐73. [DOI] [PubMed] [Google Scholar]

[dgad285-B8] 8. Wirtz PH, von Känel R, Emini L, et al. Evidence for altered hypothalamus-pituitary-adrenal axis functioning in systemic hypertension: blunted cortisol response to awakening and lower negative feedback sensitivity. Psychoneuroendocrinology. 2007;32(5):430‐436. [DOI] [PubMed] [Google Scholar]

[dgad285-B9] 9. Balbo M, Leproult R, Van Cauter E. Impact of sleep and its disturbances on hypothalamo-pituitary-adrenal axis activity. Int J Endocrinol. 2010;2010:759234. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Effects of Sleep Fragmentation and Estradiol Decline on Cortisol in a Human Experimental Model of Menopause

Aviva Y Cohn

Leilah K Grant

Margo D Nathan

Aleta Wiley

Mathena Abramson

Jessica A Harder

Sybil Crawford

Elizabeth B Klerman

Frank A J L Scheer

Ursula B Kaiser

Shadab A Rahman

Hadine Joffe

Abstract

Context

Objective

Methods

Results

Conclusion

Materials and Methods

Participants

Study Design Overview

Sleep Fragmentation Protocol

Objective Sleep Recording

Hormone Assays

Cortisol

Figure 1.

Estradiol

Data analysis

Results

Table 1.

Effect of Sleep Fragmentation Protocol on Sleep Architecture

Table 2.

Effect of Leuprolide on Estradiol Levels

Effect of Sleep Fragmentation on Bedtime Cortisol and Cortisol Awakening Response

Figure 2.

Figure 3.

Association of Polysomnography-derived Wake After Sleep Onset and Total Sleep Time With Bedtime Cortisol Levels and Cortisol Awakening Response

Figure 4.

Effect of low Estradiol State on Bedtime Cortisol Levels and Cortisol Awakening Response

Figure 5.

Figure 6.

Discussion

Conclusion

Acknowledgments

Abbreviations

Contributor Information

Funding

Disclosures

Data Availability

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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