Compared with eumenorrhoeic women, exercise-trained women with functional hypothalamic amenorrhoea (ExFHA) exhibit low heart rates (HRs) and absent reflex renin–angiotensin-system activation and augmentation of their muscle sympathetic nerve response to orthostatic stress. To test the hypothesis that their autonomic HR modulation is altered concurrently, three age-matched (pooled mean, 24±1 years; mean ± S.E.M.) groups of women were studied: active with either FHA (ExFHA; n=11) or eumenorrhoeic cycles (ExOv; n=17) and sedentary with eumenorrhoeic cycles (SedOv; n=17). Blood pressure (BP), HR and HR variability (HRV) in the frequency domain were determined during both supine rest and graded lower body negative pressure (LBNP; −10, −20 and −40 mmHg). Very low (VLF), low (LF) and high (HF) frequency power spectra (ms2) were determined and, owing to skewness, log10-transformed. LF/HF ratio and total power (VLF + LF + HF) were calculated. At baseline, HR and systolic BP (SBP) were lower (P<0.05) and HF and total power were higher (P<0.05) in ExFHA than in eumenorrhoeic women. In all groups, LBNP decreased (P<0.05) SBP, HF and total power and increased (P<0.05) HR and LF/HF ratio. However, HF and total power remained higher (P<0.05) and HR, SBP and LF/HF ratio remained lower (P<0.05) in ExFHA than in eumenorrhoeic women, in whom measures did not differ (P>0.05). At each stage, HR correlated inversely (P<0.05) with HF. In conclusion, ExFHA women demonstrate augmented vagal yet unchanged sympathetic HR modulation, both at rest and during orthostatic stress. Although the role of oestrogen deficiency is unclear, these findings are in contrast with studies reporting decreased HRV in hypoestrogenic post-menopausal women.

CLINICAL PERSPECTIVES

  • Low HRV is associated with increased risk of cardiac events in both healthy and diseased populations. Oestrogen deficiency due to menopause is associated with attenuated HRV in association with decreased vagal modulation. We examined the influence of oestrogen deficiency on HRV in physically active women with ExFHA.

  • Despite oestrogen deficiency, we report that ExFHA women demonstrate augmented, not attenuated, HRV in association with markedly elevated vagal modulation of HR.

  • Although the role of oestrogen deficiency itself is unclear, these findings suggest an exercise training–oestrogen deficiency interaction that favours augmented HRV. Such modulation may confer cardioprotective effects on ExFHA women.

INTRODUCTION

Functional hypothalamic amenorrhoea (FHA) is a reversible cause of pre-menopausal ovarian suppression. FHA is characterized by chronically low levels of circulating oestradiol that resemble those observed in post-menopausal women and in men [1,2]. The prevalence of FHA is markedly higher in active (∼1–44%) compared with sedentary (∼2–5%) women [3] and has been causally related to energy deficiency due to high energy expenditure (i.e. exercise) and insufficient caloric intake [4]. During simulated orthostatic stress, exercise-trained women with FHA (ExFHA) demonstrate lower heart rate (HR) and systolic blood pressure (SBP), augmented reflex sympathetic outflow to muscle yet absent reflex increases in plasma renin and angiotensin II compared with their eumenorrhoeic counterparts [5]. These observations suggest that cardiac autonomic regulation may also be altered in ExFHA women.

HR variability (HRV) provides both insight into cardiac vagal and sympathetic HR modulation and prognostic information [6]. Increased HRV is associated with decreased risk of cardiovascular events [6]. In contrast, low HRV attributed to elevated sympathetic modulation of HR predicts increased risk of cardiac arrhythmias post-myocardial infarction [6] and a higher risk of fatal and non-fatal cardiovascular events even in apparently healthy individuals [7,8]. Oestrogen deficiency in post-menopausal women is associated with diminished HRV as a consequence of increased sympathetic and/or decreased vagal tone [9,10]. Conversely, exercise-trained hypoestrogenic post-menopausal women exhibit higher HRV and greater cardiac vagal tone compared with their sedentary counterparts [9,10]. The influence of oestrogen deficiency on HRV in exercise-trained premenopausal women with FHA is unknown.

The purpose of the present study was to investigate the consequences of hypoestrogenaemia in physically active pre-menopausal women with FHA for HR regulation. Therefore, in oestrogen-deficient ExFHA women and oestrogen-replete exercise-trained and sedentary eumenorrhoeic women, we compared HRV both at rest and during an orthostatic hypotensive challenge [lower body negative pressure (LBNP)] to reflexively increase HR. We anticipated, from the literature [11,12], that HR would be lower in ExFHA women compared with eumenorrhoeic women, both at rest and during LBNP and hypothesized this would be due to greater parasympathetic modulation.

MATERIALS AND METHODS

Subjects

Volunteers were recruited by posters targeting both sedentary and physically active pre-menopausal women. Screening procedures included general questionnaires on exercise, eating behaviour, menstrual cycle and medical health. Eligibility criteria for the study included: (1) age 18–35 years; (2) absence of chronic illness, including diabetes, hyperprolactinaemia, poly-cystic ovarian syndrome and thyroid disease; (3) stable menstrual status over the preceding 3 months (i.e. either absence of menses or menstrual cycles between 25–35 days); (4) not taking any mediations; (5) non-smoker; (6) not currently dieting and weight stable for the preceding 3 months; (7) no history or current clinical diagnosis of eating disorders; and (8) absence of hormonal therapy for at least 6 months. The study was carried out in accordance with the Declaration of Helsinki (2008) of the World Medical Association and the study was approved by the local University and Hospital Research Ethics Boards. All volunteers provided written informed consent.

Experimental design

Volunteers were recruited consecutively over 3 years to participate in a larger cross-sectional study examining the cardiovascular consequences of oestrogen deficiency in pre-menopausal women. Fifty-seven women participated in the larger study, which comprised three smaller studies. Participants enrolled in one to three of these smaller studies. In the present paper, subject characteristic data and HR and blood pressure (BP) recorded at rest and during LBNP for exercise-trained women with eumenorrhoeic ovulatory menstrual cycles (ExOv) and ExFHA groups include, in part, data from participants that have previously been reported by our group [5,11]. The present paper reports novel data on our primary outcome variable, HRV, which was assessed at rest and during LBNP in 41 of the 57 women.

Exercise and menstrual status definitions

Volunteers were grouped according to exercise status (exercising or sedentary) and menstrual/ovulatory status (eumenorrhoeic and ovulatory or amenorrhoeic). To conform with previously published convention with respect to studies in such women, exercise status was defined as ‘sedentary’ when purposeful exercise was <2 h per week and ‘exercising’ when purposeful exercise was conducted >2 h per week [13]. Furthermore, based on literature describing ‘average’ peak aerobic capacity between 38 and 42 ml/kg/min in sedentary women [14] a cut-off of ≤40 ml/kg/min defined ‘sedentary’ status and >40 ml/kg/min reflected ‘exercising’ status. Eumenorrhoea was defined as 10–13 menstrual cycles per year. Ovulation, confirmed during the menstrual cycle prior to testing, was detected using a urinary ovulation hormone kit (Clearblue Easy ovulation test, Unipath Diagnostics). FHA in association with exercise training was defined as cessation of menses for 90 or more consecutive days [3].

Study groups

Three study groups were established: recreationally exercise trained women with FHA (ExFHA; n=11) or eumenorrhoeic ovulatory menstrual cycles (ExOv; n=17) and sedentary women with eumenorrhoeic ovulatory menstrual cycles (SedOv; n=17). To examine the effects of oestrogen deficiency in physically active women on HRV, values in amenorrhoeic physically active women with FHA were compared with those observed in eumenorrhoeic ovulatory oestrogen-replete physically active women. Sedentary eumenorrhoeic ovulatory oestrogen-replete physically active women were recruited as a ‘normal’ healthy reference group.

Subject preparation

All measures were obtained during the early follicular phase (low oestrogen and progesterone phase; days 2–6) of the menstrual cycle in eumenorrhoeic subjects and on a random day for amenorrhoeic women. Thus, we were able to compare the cardiovascular effects of cyclically low oestrogen (eumenorrhoeic women) compared with chronically low oestrogen (FHA women) levels. All tests occurred in the morning between 09:30 and 10:30 in a quiet ambient temperature room (22–24°C). Volunteers had fasted at least 2 h and abstained from alcohol for 12 h and caffeine and exercise for 24 h, prior to testing.

Anthropometric measures and body composition

Total body mass and height were determined using a physician's balance scale (Detecto). Body composition was determined using dual-energy X-ray absorptiometry (DXA; Prodigy, General Electric Lunar Corporation).

Peak aerobic capacity

On a separate study day, peak aerobic capacity (VO2 peak) was measured using a metabolic cart during a progressive treadmill test to exhaustion (Moxus Modular VO2 System, Applied Electrochemistry).

Blood sampling

Using standard venipuncture techniques, blood samples were collected at baseline for determination of serum 17β-oestradiol, progesterone, testosterone and sex-hormone-binding globulin (SHBG). On a separate day, 8-h fasted serum-free tri-iodothyronine (T3) was also assessed to provide an estimate of energy status, with low T3 levels indicating low energy status (i.e. energy deficiency) [15]. Free androgen index [(total testosterone/SHBG) × 100] was calculated to provide an estimate of androgenicity [16]. All assays were run by the Core Laboratory at the Toronto General Hospital.

Blood pressure and heart rate

Measurements of brachial SBP, diastolic BP (DBP), mean arterial BP (MAP) and HR were recorded from the left upper-arm using an automated device (Dinamap Pro 100, Critikon). Brachial BP and HR were assessed at 1-min intervals at baseline (three consecutive stable measures) and every 1 min throughout each stage of LBNP. The recordings acquired during the last 4 min of each LBNP stage were averaged to acquire a mean value for each LBNP stage. Continuous recordings of HR and BP were also acquired using lead II of an electrocardiogram (ECG) and a photoplethysmographic device on the index finger (Portapres Model-2, Finapres Medical Systems) respectively.

Heart rate variability

Frequency domain analysis of HRV was performed as described previously [17]. In brief, 7-min recordings of continuous HR intervals (R-R) were collected using lead II of an ECG during spontaneous breathing at baseline and during each stage of LBNP. The ECG signal was sampled at 1000 Hz and was stored using LabView (National Instruments) for off-line analysis. Frequency domain analysis was performed using a non-parametric method of fast-Fourier transformation (FFT). Each 7-min data set comprised 2048 data points which were divided into seven segments, each containing 512 points, with one-half overlapping of each segment. The linear trend in the data was subtracted from the data set in each segment and a Blackman–Harris window was applied to minimize spectral leakage. The power spectrum was subsequently quantified into very low (VLF: 0.001–0.05 Hz), low (LF: 0.05–0.15 Hz) and high (HF: 0.15–0.5 Hz) frequencies. Frequency domains were determined in absolute units (ms2) and as per Task Force recommendations [18], HF and LF power were both transformed (log10) and normalized [%; LFnu=LF power/(total power–VLF power) × 100 and HFnu=HF power/(total power–VLF power) × 100]. HF power is considered an index of vagal HR modulation and LF power is considered an index of primarily sympathetic modulation, with some contribution from the efferent vagus [18]. The LF/HF ratio has been proposed as a rough estimate of cardiac sympatho-vagal balance [18]. For all data sets, only stationary time series with ≤5% arrhythmia or artefact were used for analyses.

LBNP

With subjects in the supine position, the lower body was encased in a custom-built chamber that was sealed at the level of the iliac crest [19]. The chamber was attached to a vacuum source to effect reductions in pressure. HR, BP, respiratory excursions and HRV were continuously recorded at baseline and during each 8-min stage of LBNP (−10, −20 and −40 mmHg). Each LBNP stage was followed by 5 min of recovery (i.e. no LBNP). LBNP was terminated if SBP declined to <80 mmHg, pallor was observed and/or subjective feelings of nausea, dizziness and/or light-headedness were reported.

Statistics

All data sets were tested for non-normality, homogeneity of variance and outliers. Consistent with previous literature [18], raw HRV data were non-normally distributed. Thus, both normalized and log10-converted LF and HF raw data were calculated. At baseline, between-group differences were detected using one-way ANOVA and when a significant main (fixed) effect was observed, within-group and between-group analyses of responses to LBNP, including change (∆) in measures compared with baseline, were determined using mixed-model ANOVA, using Group as the between-group factor. When significant within-group and between-group differences were detected, Bonferroni methods were used to determine where the significant differences existed. When assumptions of sphericity were violated, the Greenhouse–Geisser correction was used. Using pooled data, Pearson's correlational analyses were used to determine significant linear-independent associations between HRV indices and variables of interest. Data were analysed using packaged software (SPSS version 20; SPSS Inc.). All data are presented as means ± S.E.M. A significance level of P<0.05 was used to detect the differences for statistical procedures.

RESULTS

Subject characteristics

Groups did not differ (P>0.05) in age, height, weight or body composition (Table 1). Sedentary women were significantly less aerobically conditioned (P<0.001) compared with exercising women. Serum measures of 17β-oestradiol (pmol/l) did not differ (P>0.05) between ovulatory groups. In contrast, 17β-oestradiol was lower (P<0.05) in ExFHA compared with ExOv only. Progesterone was similar (P>0.05) between groups (1.8±0.7 nmol/l; pooled mean). Free T3 (pmol/l) was significantly lower (P<0.05) in ExFHA compared with ExOv and sedentary women with eumenorrhoeic ovulatory menstrual cycles (SedOv) women. Testosterone and free androgen index (FAI) did not differ (P>0.05) between groups. SHBG was lower (P<0.05) in SedOv compared with ExOv and ExFHA women.

Table 1
Demographic and anthropometric measures of the study groups

Values are means ± S.E.M. Abbreviations: BMI, body mass index; FAI, free androgen index. *ExFHA compared with ExOv. †ExFHA compared with ExOv and SedOv. ‡SedOv compared with ExOv and ExFHA.

SedOv (n=17)ExOv (n=17)ExFHA (n=11)P-value (main effect)
Age (years) 23.5±0.6 23.5±1.2 25.4±1.1 0.312 
Height (m) 1.65±1.5 1.66±1.5 1.67±1.5 0.400 
Weight (kg) 58.6±1.5 58.0±1.9 58.7±2.3 0.953 
BMI (kg/m221.7±0.5 20.9±0.5 20.8±0.8 0.438 
Body fat (%) 28.7±2.3 24.7±2.0 21.8±2.2 0.118 
Fat-free mass (kg) 38.8±1.4 42.0±1.6 44.1±1.3 0.092 
Oestradiol (pmol/l) 130.9±10.9 148.7±19.2 87.8±14.7* 0.007 
Progesterone (nmol/l) 1.7±0.2 1.8±0.2 1.7±0.2 0.895 
T3 (pmol/l) 4.2±0.2 4.3±0.1 3.6±0.6 <0.001 
Testosterone (nmol/l) 2.4±0.2 2.1±0.2 2.4±0.2 0.371 
SHBG (nmol/l) 40.5±2.7 52.9±1.9 51.6±4.1 0.020 
FAI (au) 0.06±0.02 0.05±0.01 0.05±0.01 0.137 
VO2 peak (ml/kg/min) 38.9±0.8 46.6±1.3 47.8±2.0 <0.001 
SedOv (n=17)ExOv (n=17)ExFHA (n=11)P-value (main effect)
Age (years) 23.5±0.6 23.5±1.2 25.4±1.1 0.312 
Height (m) 1.65±1.5 1.66±1.5 1.67±1.5 0.400 
Weight (kg) 58.6±1.5 58.0±1.9 58.7±2.3 0.953 
BMI (kg/m221.7±0.5 20.9±0.5 20.8±0.8 0.438 
Body fat (%) 28.7±2.3 24.7±2.0 21.8±2.2 0.118 
Fat-free mass (kg) 38.8±1.4 42.0±1.6 44.1±1.3 0.092 
Oestradiol (pmol/l) 130.9±10.9 148.7±19.2 87.8±14.7* 0.007 
Progesterone (nmol/l) 1.7±0.2 1.8±0.2 1.7±0.2 0.895 
T3 (pmol/l) 4.2±0.2 4.3±0.1 3.6±0.6 <0.001 
Testosterone (nmol/l) 2.4±0.2 2.1±0.2 2.4±0.2 0.371 
SHBG (nmol/l) 40.5±2.7 52.9±1.9 51.6±4.1 0.020 
FAI (au) 0.06±0.02 0.05±0.01 0.05±0.01 0.137 
VO2 peak (ml/kg/min) 38.9±0.8 46.6±1.3 47.8±2.0 <0.001 

Blood pressure and heart rate

Baseline values

Between groups, HR and SBP were lower (P<0.05) in ExFHA women than in SedOv and ExOv women, in whom HR and SBP did not differ (P>0.05; Table 2). Baseline DBP was similar (P>0.05) between the groups.

Table 2
BP and HR responses to graded LBNP

Values are means ± S.E.M. Abbreviation: Ba, baseline. *LBNP main effect for HR, SBP, DBP and MAP within each group, P<0.001. †Significantly different from baseline within groups, P<0.05. ‡ExFHA compared with SedOv and ExOv within condition, P<0.05.

HRSBPDBPMAP
SedOv* 
Ba 61±2 102±2 59±1 74±1 
−10 mmHg 61±2 98±2 55±1 69±1 
−20 mmHg 65±3 96±1 53±1 67±2 
−40 mmHg 80±3 94±2 51±2 66±2 
ExOv* 
Ba 57±2 105±2 61±1 76±1 
−10 mmHg 58±2 102±2 57±2 72±2 
−20 mmHg 62±3 100±2 54±2 70±2 
−40 mmHg 72±3 98±1 53±2 68±2 
ExFHA* 
Ba 47±2 94±2 58±2 70±2 
−10 mmHg 48±2 93±2 56±2 68±2 
−20 mmHg 49±2 92±2 53±2 66±2 
−40 mmHg 60±2†‡ 89±2†‡ 51±2 64±2 
HRSBPDBPMAP
SedOv* 
Ba 61±2 102±2 59±1 74±1 
−10 mmHg 61±2 98±2 55±1 69±1 
−20 mmHg 65±3 96±1 53±1 67±2 
−40 mmHg 80±3 94±2 51±2 66±2 
ExOv* 
Ba 57±2 105±2 61±1 76±1 
−10 mmHg 58±2 102±2 57±2 72±2 
−20 mmHg 62±3 100±2 54±2 70±2 
−40 mmHg 72±3 98±1 53±2 68±2 
ExFHA* 
Ba 47±2 94±2 58±2 70±2 
−10 mmHg 48±2 93±2 56±2 68±2 
−20 mmHg 49±2 92±2 53±2 66±2 
−40 mmHg 60±2†‡ 89±2†‡ 51±2 64±2 

LBNP responses

Within groups, graded LBNP elicited a significant increase (P<0.05) in HR and a decrease (P<0.05) in SBP and DBP. Between groups, analyses showed that HR and SBP values during LBNP remained significantly lower (P<0.05) in ExFHA compared with SedOv and ExOv women. DBP values during LBNP did not differ (P>0.05, main effect) between the groups. No between-group differences for BP and HR values were detected during graded LBNP for SedOv and ExOv women (P>0.05). Δ values (not shown) for all HR and BP responses did not differ (P>0.05) between all groups.

Heart rate variability

Baseline values

Complete HRV data were unavailable in one SedOv woman whose study was terminated when pre-syncopal symptoms were evoked by −20 mmHg LBNP. ExFHA women demonstrated higher (P<0.05; main effect) baseline HF (ms2), total HRV and HFlog10 compared with SedOv and ExOv women (Table 3). The LF/HF ratio trended lower (P=0.095) in ExFHA women than ovulatory women. In contrast, groups did not differ (P>0.05) with respect to baseline VLF (ms2), LF (ms2), LF/HF ratio, LFnu, HFnu and LFlog10. HRV did not differ (P>0.05) between ExOv and SedOv women.

Table 3
Baseline HRV for the study groups

Values are means ± S.E.M. *ExFHA compared with SedOv and ExOv, P<0.05. †ExFHA compared with SedOv, P<0.05.

SedOv (n=17)ExOv (n=17)ExFHA (n=11)p-value (main effect)
Absolute (ms2
LF 686±135 1024±265 1023±165 0.398 
HF 837±122 948±130 2364±757* 0.012 
VLF 818±115 1160±179 1705±549 0.114 
LF + HF 1524±220 1971±341 3386±894 0.034 
Total (LF + HF + VLF) 2347±253 3137±349 5092±1310* 0.021 
LF/HF  0.9±0.1  1.1±0.2  0.6±0.01 0.095 
Normalized (%) 
LF (LF/[LF+HF] × 100) 44±3 46±5 34±4 0.129 
HF (HF/[LF+HF] × 100) 56±3 54±5 66±4 0.129 
Transformed (log10
LF  2.7±0.1  2.8±.01  2.9±0.1 0.516 
HF  2.8±0.1  2.9±0.1  3.2±0.1* 0.022 
SedOv (n=17)ExOv (n=17)ExFHA (n=11)p-value (main effect)
Absolute (ms2
LF 686±135 1024±265 1023±165 0.398 
HF 837±122 948±130 2364±757* 0.012 
VLF 818±115 1160±179 1705±549 0.114 
LF + HF 1524±220 1971±341 3386±894 0.034 
Total (LF + HF + VLF) 2347±253 3137±349 5092±1310* 0.021 
LF/HF  0.9±0.1  1.1±0.2  0.6±0.01 0.095 
Normalized (%) 
LF (LF/[LF+HF] × 100) 44±3 46±5 34±4 0.129 
HF (HF/[LF+HF] × 100) 56±3 54±5 66±4 0.129 
Transformed (log10
LF  2.7±0.1  2.8±.01  2.9±0.1 0.516 
HF  2.8±0.1  2.9±0.1  3.2±0.1* 0.022 

LBNP responses

Within-groups, LBNP elicited significant reductions in total HRV [F(1.92, 82.66)=7.175, P=0.002; main effect], HFnu [F(2.31, 99.29)=46.54, P<0.001] and HFlog10 [F(1.63, 70.18)=72.411, P<0.001; Figure 1]. In contrast, LF/HF ratio [F(1.42, 61.07)=30.156, P<0.001] and LFnu [F(2.29, 102.92)=44.239, P<0.001] were increased by LBNP. VLF (ms2), LF (ms2) and LFlog10 (ms2) were unaltered (P>0.05) by LBNP. No LBNP × Group interactions (P>0.05) were detected. Between groups, ExFHA women demonstrated significantly higher total HRV (P=0.001; main effect), HFnu (P<0.001; main effect) and HFlog10 (P<0.001; main effect) at each LBNP stage compared with SedOv and ExOv women. Conversely, LF/HF ratio was significantly lower (P=0.023; main effect) at −20 mmHg and −40 mmHg in ExFHA compared with SedOv women only. HRV did not differ (P>0.05) between SedOv and ExOv women at any LBNP stage. Δ HRV responses to LBNP did not differ (P>0.05) between the groups.

Frequency domain indices of HRV at baseline and during graded LBNP in SedOv (closed circles), ExOv (closed squares) and ExFHA (open triangles) women
Figure 1
Frequency domain indices of HRV at baseline and during graded LBNP in SedOv (closed circles), ExOv (closed squares) and ExFHA (open triangles) women

HRV indices include: HFlog10 (A), LFlog10 (B), total HRV (C) and LF/HF (D). *ExFHA compared with SedOv and ExOv, P<0.05. §ExFHA compared with ExOv, P<0.05. L × G, LBNP × Group interaction.

Figure 1
Frequency domain indices of HRV at baseline and during graded LBNP in SedOv (closed circles), ExOv (closed squares) and ExFHA (open triangles) women

HRV indices include: HFlog10 (A), LFlog10 (B), total HRV (C) and LF/HF (D). *ExFHA compared with SedOv and ExOv, P<0.05. §ExFHA compared with ExOv, P<0.05. L × G, LBNP × Group interaction.

Respiration rate

Respiration rate did not differ (P>0.05; main effect) between groups at baseline (14.2±0.4 breaths/min; overall baseline mean ± S.E.M.), neither was it altered by LBNP (P>0.05, main effect).

Correlates of HR and HRV

Resting HR was positively associated with 17β-oestradiol (r=0.517, P=0.001) and T3 (r=0.611, P<0.001). In contrast, baseline measures of LFlog10 (r=−0.028), HFlog10 (r=−0.299) and total HRV (r=−0.164) were not associated (P>0.05) with serum measures of 17β-oestradiol. No resting measure for HRV correlated with T3 (all P>0.05).

DISCUSSION

To our knowledge, this is the first study to evaluate autonomic HR modulation using frequency domain analysis of HRV in oestrogen-deficient exercise-trained pre-menopausal women with FHA. We report that compared with age-matched eumenorrhoeic sedentary and exercise-trained oestrogen-replete women, exercise-trained oestrogen-deficient pre-menopausal women have lower HR both at rest and during orthostatic stress. LBNP reflexively increased HR in all groups in association with lowered HF, unaltered LF and increased LF/HF ratio. However, hypoestrogenic exercise trained pre-menopausal women demonstrated higher HF and total power values at rest and during orthostatic stress compared with their exercise-trained and sedentary eumenorrhoeic peers. LF/HF ratio was also significantly lower in amenorrhoeic than eumenorrhoeic sedentary women during LBNP. Collectively, these observations are consistent with the concept that otherwise healthy hypoestrogenic exercise-trained pre-menopausal women have augmented vagal HR modulation, resulting in greater total HRV spectral power.

Oestrogen and autonomic HR modulation

Several lines of evidence support the concept that oestrogen modulates HR and cardiac autonomic tone. For example, pre-menopausal women demonstrate higher basal cardiac vagal tone compared with age-matched males [20,21] and post-menopausal women [21]. Oestrogen therapy lowers resting HR in normotensive post-menopausal women [22] and increases vagal and/or decreases sympathetic HR modulation at rest in most [20,21], but not all [23], post-menopausal women. The effects of fluctuating oestrogen levels across menstrual cycle on HRV are less clear, probably in part due to differences in the timing of data collection across the cycle. Notwithstanding, studies variably report greater [24,25] and unaltered [26] HRV during the follicular (increasing oestrogen, low progesterone) compared with the mid-luteal (high oestrogen and progesterone) phase. In animals, 17β-oestradiol administration increases parasympathetic and decreases sympathetic modulation of HR in both male and ovariectomized rodents [27]. The expression of significant populations of both oestrogen receptor-α and -β on cell bodies, axons and terminals of autonomic regulatory nuclei throughout the neuraxis is thought to explain in part the autonomic effects of oestrogen [28].

In contrast with the literature in post-menopausal women, we report that, despite hypoestrogenaemia, LF HRV did not differ between the three study groups, yet vagal modulation of HR was enhanced both at rest and during LBNP in ExFHA women. Furthermore, the LF/HF ratio was lower during −20 and −40 mmHg LBNP in ExFHA compared with sedentary eumenorrhoeic women, suggesting that their reflex sympathetic HR response to this stimulus may also be attenuated. An alternative interpretation is that the parasympathetic response was reflected in both the numerator and the denominator of this ratio. It has been demonstrated that LF is not purely an index of cardiac sympathetic modulation, rather it is a marker of both sympathetic and vagal HR modulation, including modulation by the arterial baroreflex [29]. The supine position, as utilized in the present study, is also associated with greater vagal values compared with the seated or standing position [30]. Thus, it is possible that, in the present study, supine LF HRV, and indeed LF/HF, was primarily influenced by parasympathetic modulation. Because of this complexity, HF rather than LF was our principal focus of investigation in this experiment.

Collectively, low resting HR and augmented HRV in ExFHA women at rest and during LBNP suggests that these findings are not a direct consequence of oestrogen deficiency. In the light of the aetiology of FHA in physically active women, alternative factors contributing to the elevated parasympathetic modulation of HR in ExFHA may include energy deficiency (i.e. caloric deficit), aerobic exercise training and low brain and circulating angiotensin II concentrations, all of which are independently associated with augmented vagal modulation of HR [9,10,31,32]. The possible contribution of these three factors to the observed enhanced HRV in ExFHA women are considered below.

Energy deficiency and HR modulation

Amenorrhoea in weight-stable active women is causally linked to mild negative energy balance in association with increased energy expenditure that is frequently combined with subtle deficits in caloric intake [4]. In response to energy deficiency, circulating T3 levels are decreased in ExFHA women [33]. In accordance with this, we report low T3 in ExFHA women compared with eumenorrhoeic women. T3 is known to exert positive modulatory effects on HR by binding primarily to thyroid receptor-α expressed in the heart and the vasculature, eliciting genomic and non-genomic actions that influence gene transcription and peripheral haemodynamics [34]. As previously reported by our group [33], we observed a strong positive association between circulating levels of T3 and HR. In contrast, we did not observe a correlation between T3 and any index of HRV. This observation is perhaps not too surprising since the contemporary literature concerning thyroid hormones and HRV has yet to achieve consensus. For example, the influence of the parasympathetic nervous system on HR is reported as being reduced in both hypo- and hyper-thyroidism, with pharmacological restoration of euthyroid status eliciting increases in cardiac vagal tone [35,36]. Conversely, in both human and animal studies, low T3 elicited by caloric restriction alone has been correlated positively with HRV due to increased HF and/or decreased LF power [34].

Exercise training and HR modulation

Aerobic conditioning in humans is associated with resting bradycardia and increased HRV with greater resting parasympathetic and less sympathetic efferent HR modulation [9,10]. Alterations in the intrinsic sino-atrial node pacemaker [37] and neurotransmission at the level of the nucleus tractus solitarius [38] have been implicated in these changes. In the present study, we report similar cardio-respiratory fitness in our exercising groups yet lower HR and higher vagal modulation of HR both at rest and during LBNP in ExFHA compared with ExOv women. In active young women, high-volume and high-intensity exercise training elicits greater vagal modulation of HR at rest and during postural stress compared with women undertaking low-intensity and low-volume exercise training [39], and high-intensity exercise training leads to greater cardiorespiratory fitness than moderate- or low-intensity exercise training [40]. With the two active groups having similar fitness, it is therefore unlikely that exercise training intensity itself can account for the augmented vagal modulation of HR in ExFHA women. Interestingly, the present finding is consistent with reports of augmented cardiac vagal modulation of HR in aerobically trained ovariectomized compared with intact rats [41,42], suggesting that hypoestrogenaemia may modulate exercise-training-induced effects on parasympathetic HR modulation. Examination of such postulates awaits delineation.

Angiotensin II and HR modulation

Recently, our group reported that, compared with oestrogen replete ExOv women, ExFHA women demonstrate an absence of reflex activation of circulating renin and angiotensin II in response to orthostatic stress [5]. Substantial evidence links the renin–angiotensin–aldosterone system and the sympathetic nervous system [32,43,44]. Interactions can occur at central sites, such as in the rostral ventrolateral medulla and hypothalamus and at the sympathetic pre-junctional angiotensin II type 1 receptor, which facilitates noradrenaline (norepinephrine) release from the sympathetic nerve terminal [32,43,44]. Blockade of the angiotensin II type 1 receptor decreases sympathetic modulation of HR in experimental heart failure [44] and administration of angiotensin-converting enzyme inhibitors increases vagal modulation of HR in patients after acute myocardial infarction [43]. Thus, angiotensin II exerts both sympatho-stimulatory and vagal-inhibitory effects on HR modulation [32]. Of note, angiotensin II infusion has been reported to elicit sex-specific cardiac autonomic responses, with healthy young men exhibiting a shift toward sympatho-vagal imbalance due to withdrawal of parasympathetic activity and healthy young women demonstrating unaltered sympatho-vagal balance [45]. Whether resistance to the vagolytic effects of angiotensin II infusion is diminished in functionally hypoestrogenaemic women is unknown. However, our previous finding of significantly lower circulating angiotensin II concentrations at −20 and −40 mmHg in ExFHA (2.1±0.3 and 2.0±0.4 pmol/l respectively) compared with ExOv women (5.3±1.4 and 8.6±1.9 pmol/l respectively) [5] suggests that low circulating angiotensin II in ExFHA women during orthostatic stress may contribute to their augmented cardiac parasympathetic tone during LBNP.

Limitations

Respiratory sinus arrhythmia or the HF (vagal) component of HRV, is influenced by both the depth and the frequency of respiration [46]. Despite having similar breathing frequencies (∼14 breaths/min) among our study groups; it is possible that women with ExFHA may have different tidal volumes, which were not recorded. Owing to the low prevalence (∼2–5%) [3] and the psychogenic- and/or weight-loss-related origins of FHA in sedentary women [47], our investigations did not extend to the effects of pre-menopausal oestrogen deficiency on HRV in such women. Therefore, our study findings are limited to the effects of oestrogen deficiency combined with exercise training on HRV.

Conclusion

Oestrogen is assumed to elicit cardioprotective effects on the autonomic nervous system, including elevated HRV in association with increased vagal and decreased sympathetic modulation of HR [21]. In contrast with this concept, we report that physically active oestrogen-deficient pre-menopausal women with FHA demonstrate elevated HRV as a consequence of increased vagal HR modulation both at rest and during graded orthostatic stress. Furthermore, HRV is elevated in ExFHA women above that observed in fitness-matched oestrogen-replete women. Although the influence of oestrogen deficiency itself on cardiac autonomic modulation in ExFHA women is unclear, these findings suggest complex interactions between competing factors may oppose the cardiac autonomic-modulatory effects of oestrogen deficiency. In the light of the aetiology of FHA in active women, such factors probably include low circulating levels of T3, angiotensin II and exercise training–oestrogen interactions.

AUTHOR CONTRIBUTION

Emma O'Donnell conducted the study, collected data, ran statistics, interpreted data, drafted manuscript, figures and tables and proofread the manuscript. Jack Goodman interpreted the data and edited and proofread the manuscript. Beverly Morris conducted the study and proofread the manuscript. John Floras interpreted the data and edited and proofread the manuscript. Paula Harvey obtained funding, prepared study design and edited and proofread the manuscript. All authors read and approved the final paper.

We acknowledge and thank all the members of the Clinical Cardiovascular Physiology Laboratory at the Toronto General Hospital for their technical assistance and collaboration. We also thank each of our participants for their time and efforts. John S. Floras is a Canada Research Chair in Integrative Cardiovascular Biology.

FUNDING

This work was supported by a Pfizer Cardiovascular Independent Research Award [grant number NRA 3840028] to P.J.H.

Abbreviations

     
  • BP

    blood pressure

  •  
  • DBP

    diastolic blood pressure

  •  
  • ECG

    electrocardiogram

  •  
  • ExFHA

    exercise-trained women with functional hypothalamic amenorrhoea

  •  
  • ExOv

    exercise-trained women with eumenorrhoeic ovulatory menstrual cycles

  •  
  • FHA

    functional hypothalamic amenorrhoea

  •  
  • HF

    high frequency

  •  
  • HR

    heart rate

  •  
  • HRV

    heart rate variability

  •  
  • LBNP

    lower body negative pressure

  •  
  • LF

    low frequency

  •  
  • MAP

    mean arterial blood pressure

  •  
  • SBP

    systolic blood pressure

  •  
  • SedOv

    sedentary women with eumenorrhoeic ovulatory menstrual cycles

  •  
  • SHBG

    sex-hormone-binding globulin

  •  
  • T3

    tri-iodothyronine

  •  
  • VLF

    very low frequency

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