Psychoneuroendocrinology 26 (2001) 225–240

www.elsevier.com/locate/psyneuen

Gender differences in age-related changes in

HPA axis reactivity
a,*
Teresa E. Seeman , Burton Singer b, Charles W. Wilkinson c,
Bruce McEwen d
a
Division of Geriatrics, UCLA School of Medicine, 10945 Le Conte Ave, Suite 2339, Los Angeles,
CA 90095-1687, USA
b
Office of Population Research, Princeton University, Princeton, NJ, USA
c
Dept. of Psychiatry and Behavioral Sciences, University of Washington and Geriatric Research,
Education and Clinical Center, VA Puget Sound Health Care System, Seattle, WA, USA
d
Laboratory of Neuroendocrinology, Rockefeller University, New York, NY, USA

Received 10 August 1999; accepted 25 July 2000

Abstract

Possible differences between men and women in age-related patterns of hypothalamic–pitu-

itary–adrenal (HPA) axis response to challenge were examined to test the hypothesis that
women show greater age-related increase in HPA axis reactivity to challenge. Twenty-six
younger subjects, 9 men and 17 women, ages 22–26 and 14 older subjects, 7 men and 7
women, ages 67–88 participated in the study. Patterns of change in salivary “free” cortisol
were measured in response to a standardized, 30-minute cognitive challenge, administered
individually to each subject beginning at 1600 h. Consistent with previous research, there was
a significant main effect for age with respect to baseline cortisol: older age was associated
with higher baseline cortisol (P=⬍0.001). Results also provide support for the hypothesized
age-by-gender interaction with respect to patterns of response to challenge. There was a sig-
nificant interaction with respect to maximum percentage increase over baseline (P⬍0.002):
among younger adults, the men exhibited greater increases whereas among the older adults,
the women exhibited greater increases. A similar, though only marginally significant pattern
was seen for total area under the response curve (P=0.07). Repeated measures ANOVA con-
firmed the gender-by-age differences in the patterns of response (P=0.01 for time*age*gender
interaction).  2001 Elsevier Science Ltd. All rights reserved.

* Corresponding author.
E-mail address: tseeman@mednet.ucla.edu (T.E. Seeman).

0306-4530/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 6 - 4 5 3 0 ( 0 0 ) 0 0 0 4 3 - 3
226 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

Keywords: Age differences; Gender differences; HPA axis reactivity; Cognitive challenge

1. Introduction

Gender differences in morbidity and mortality remain a topic of considerable

research interest. Although women enjoy greater longevity (Verbrugge and Wingard,
1987), their relative protection against major causes of morbidity and mortality such
as heart disease is greatly reduced post-menopause (Greendale et al., 1999). Though
there has been considerable research directed towards understanding the sources of
observed gender differences in health and longevity, much remains to be elucidated.
Here, we examine the question of possible gender differences in age-related changes
in hypothalamic–pituitary–adrenal (HPA) axis reactivity to challenge. Such gender
differences are of interest as potential contributors to gender differences in health
risks and, in particular, to the known reduction in women’s relative protection against
heart disease and stroke post-menopause. Specifically, we examine the hypothesis
that women exhibit greater age-related increase in HPA axis reactivity, resulting in
greater exposure to elevated levels of cortisol.
Data on patterns of diurnal, plasma cortisol activity suggest an age-related increase
in basal cortisol secretion (Deuschle et al., 1997; van Cauter et al., 1996). Signifi-
cantly, recent analyses comparing age-related changes for men versus women have
suggested that women experience larger age-related increases in cortisol secretion,
with postmenopausal women exhibiting shorter nocturnal nadirs, higher morning
acrophase and higher overall mean 24-hr cortisols (van Cauter et al., 1996). Studies
also suggest that women may experience greater age-related increase in HPA axis
response to challenge. At younger ages, studies show either greater cortisol response
among men (Collins and Frankenhaeuser, 1978; Kirschbaum et al., 1992b) or no
significant gender differences (Gallucci et al., 1993; Stoney et al., 1987; Streeten et
al., 1984). A number of studies of older men and postmenopausal women, however,
show intriguing patterns of greater HPA axis reactivity among women in response
to CRH challenge (Greenspan et al., 1993; Heuser et al., 1994; Luisi et al., 1998)
and a trend towards greater response to physostigmine, a centrally active cholinester-
ase inhibitor (Peskind et al., 1995). Wilkinson et al. (1997) have also reported that
postmenopausal women exhibit less feedback inhibition (i.e., blunted ACTH decline)
in response to cortisol infusion, post-metyrapone administration, compared with
younger, pre-menopausal women and with older men. Patterns of response to psycho-
logical challenge have to date shown a more mixed picture. One study has reported
greater ACTH and cortisol response among men in response to a public-speaking
challenge (Kudielka et al., 1998). By contrast, we found a pattern of greater ACTH
and cortisol in older women as compared with older men in response to a “driving
simulation” challenge (Seeman et al., 1995).
Several lines of evidence suggest the possible role of reproductive hormones such
as estrogen in this apparent age-related shift towards greater relative HPA axis reac-
tivity among postmenopausal women. First, variations in estrogen have been linked
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 227

to altered reactivity — periods of higher estrogen being associated with reduced

blood pressure (von Eiff et al., 1971), cardiovascular (Sita and Miller, 1996) and
HPA reactivity (Bonen et al., 1991; Marinari et al., 1976) though there are also data
suggesting that this reduced reactivity may be lost in the presence of higher pro-
gesterone levels (Kirschbaum et al., 1999). Second, and more important for the postu-
lated age-related increase in HPA reactivity in postmenopausal women, hormone
replacement therapy (HRT) has been associated with reductions in blood pressure
responses to challenge (Stoney, 1992) and with a blunting of cortisol responses to
cognitive and physical stress tests in postmenopausal women exposed short-term (six
weeks) to 17β-estradiol (Lindheim et al. 1992, 1994).
To date, however, no study has directly tested for a gender difference in age-
related changes in patterns of HPA reactivity. Rather, studies have generally exam-
ined gender differences either in older adults or in younger subjects but rarely have
such differences been examined for both age groups simultaneously. Also, no studies
have directly tested for an age-by-gender interaction with respect to patterns of HPA
axis reactivity to challenge. The study reported here was designed to assess age and
gender differences in patterns of HPA axis response to a single, structured cognitive
challenge. Specifically, we sought to test the hypothesis that there would be an inter-
action such that among younger adults the men would be more reactive while among
the older adults the reverse would be true with women exhibiting greater cortisol
reactivity to the cognitive challenge.

2. Methods

2.1. Study participants

Younger and older men and women were selected for this study on the basis of
age, gender and health status. Older subjects were sampled from an existing pool of
older, healthy adults maintained by the University of Southern California’s Alzhei-
mer’s Disease Research Center as control subjects in their studies of Alzheimer’s
disease. Younger subjects were sampled from student volunteers from a local college.
A phone interview was administered to all potential subjects to identify those who
met age criteria (i.e., 20–40 for the younger sample, 65+ for the older sample) and
health criteria (i.e., no known diabetes, heart disease, hypertension or endocrine dis-
order, body mass index=20–29 based on self-reported height and weight, and no
reported use of medications). Criteria for study eligibility were designed to ensure
safety and to eliminate subjects whose health status might affect their pattern of
HPA axis reactivity. The final sample included 9 younger men and 17 younger
women (ages 22–36) as well as 7 older men and 7 older women (ages 67–88). The
study was approved by the USC Human Investigation committee and written infor-
med consent was obtained from each subject prior to the challenge session.
228 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

2.2. Challenge protocol

Eligible subjects participated in a single data collection session, from 1400–1830

h. Subjects arrived at the research offices at 1400 h, the study protocol was explained
and informed consent was obtained. Subjects were then to remain seated and to rest
until 1600 h; during this time, they could read and/or listen to music. To control for
the circadian rhythm of cortisol (van Cauter et al., 1996), all challenge sessions
began at 1600 h. The challenge protocol consisted of a battery of cognitive tests
which have been shown to elicit significant increases in HPA and SNS activity
(Albert et al., unpublished data; Barnes et al., 1982; Fibiger et al., 1984; Moyer et
al., 1994; Stoney et al., 1988; Williams et al., 1982). The series of tasks was adminis-
tered in a fast-paced, time-limited manner and lasted 30 minutes. Tasks, in order of
administration, included a paired-associates task from the Weschler Memory Scale-
R, the digit symbol and Stroop Color Word tasks and delayed recall of the paired-
associates. Challenging aspects of the protocol were emphasized by repeated
reminders of time remaining and by interruptions to correct mistakes. This protocol
had been pilot-tested and shown to elicit HPA axis response (Seeman, unpublished
data). All subjects fasted after 1400 h; no smoking or alcohol consumption was
permitted from noon until the end of the challenge session and subjects were permit-
ted to drink only water throughout the session. Subjects were seated upright through-
out the challenge protocol.

2.3. HPA axis reactivity

Salivary cortisol samples were used to assess changes in plasma free cortisol levels
in response to the challenge. Salivary cortisol has been shown to be a reliable, non-
invasive method of assessing plasma cortisol levels (Kirschbaum and Hellhammer,
1989; Reid et al., 1992) and has the further advantage of measuring plasma free
cortisol levels, hypothesized to be the more biologically-active form of plasma cor-
tisol (Elkins, 1990). Salivary cortisol provides a measure of non-protein bound, “free”
cortisol levels. Salivary “free” cortisol levels track closely with plasma levels, show-
ing a 1–2 min lag and have been previously used to monitor cortisol activity in both
ambulatory and laboratory challenge paradigms (Kirschbaum et al., 1992a). Saliva
samples were collected with salivettes (Sarstedt, Rommelsdorf, Germany), plastic
vials containing small cotton dental rolls. Subjects are simply asked to place the
cotton roll in their mouth until it is saturated with saliva; the cotton is then replaced
in the vial. Vials were stored, frozen at ⫺80C until lab analysis.
The first, baseline salivary samples were obtained at 1550 h and 1600 h. The 30-
minute cognitive challenge protocol then began. Additional salivary samples were
obtained at the mid-point of the challenge and immediately following the final test.
The subject was then allowed to read and/or listen to music for two hours. During
this time, five additional saliva samples were obtained (at 15, 30, 60, 90 and 120
minutes post-challenge) to assess patterns of recovery post-challenge.
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 229

2.4. Biochemical assays

Assays for salivary cortisol were performed by Dr. Charles Wilkinson’s laboratory
using a 125–I cortisol kit from Pantex (Santa Monica, CA), modified for use with
saliva samples. Salivettes were centrifuged at 4°C at approximately 1800 g for 30
minutes; 100 µl samples of the supernatant are assayed in duplicate. The sensitivity
of the assay for saliva samples is 0.02 µg/dl. The intra-assay coefficient of variation
was 6.0% and the inter-assay coefficient of variation was 6.9%.

2.5. Covariates

Education, measured in terms of the highest year of schooling completed, was

considered as a potential covariate because of its possible influence on the level of
challenge experienced in completing the cognitive tasks. Depressive symptomatology
was also considered as a potential confounder since previous research has suggested
that depression can be associated with higher cortisol (Gold et al., 1996) and
depression is also more commonly seen in women (Pearlstein et al., 1997). The
Center for Epidemiologic Studies Depression (CESD) scale was used to assess
depression (Radloff, 1977); subjects were classified as depressed or not based on
the standard cut-point of 16 or more on the CESD items. Nine subjects had scores
of 16 or more on the CESD: 3 young men, 5 young women, and 1 older woman.
Other factors initially considered as potential confounders included oral contracep-
tive (OC) use among the younger women and smoking status. Among the pre-meno-
pausal women, 6 out of 17 reported use of oral contraceptives (OC); among the post-
menopausal women, none was currently using hormone-replacement therapy (HRT).
Smoking status was also considered since previous research had suggested that nic-
otine exposure can increase cortisol activity (Wust et al., 1992). Among study parti-
cipants, three young subjects (2 males, 1 female) and two older subjects (1 male, 1
female) reported occasional smoking; all indicated that they had complied with our
request to refrain from smoking the day of the challenge session.

2.6. Analyses

Hypothesized age-by-gender differences in salivary cortisol responses were exam-

ined using ANOVA models with age-by-gender interaction terms. Group differences
were also examined using medians, rather than means, as the measure of central
tendency. In all cases, results were unchanged from those based on means. All mod-
els included depression as a covariate because it was found to be differentially dis-
tributed by age (younger subjects reported more depression) and was associated with
higher baseline cortisol (see Results). Since previous reports have suggested a poss-
ible impact of OC use on HPA axis reactivity (Bonen et al., 1991; Kirschbaum et
al., 1995), OC status was also considered as a potential confounder. Comparison of
baseline cortisol and responses to challenge for the OC and non-OC users among
the pre-menopausal women revealed no differences in either baseline cortisol or
responses to the challenge. Furthermore, comparisons of analyses with and without
230 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

the OC users indicated that the findings were unaltered. Thus, analyses reported here
include the OC users. A parallel set of analyses were run, comparing cortisol data
for smokers and non-smokers. Again, results indicated that the smokers (who had
refrained from smoking the day of the challenge session) did not differ from non-
smokers with respect to their cortisol values and that analyses excluding the smokers
did not result in altered findings. Based on these findings, results are reported for
models which include all subjects, controlling only for depression since that was the
only factor that demonstrated a relationship with both age and cortisol activity and
exclusion of those classified as depressed from the analyses did not alter the findings.
Outcomes examined included average baseline cortisol levels, maximum increases
in cortisol in response to the challenge and total area under the response curve
(AUC). Two measures of area under the curve were calculated according to the
trapezoid formula — one included baseline level in the total score, the other sub-
tracted out baseline to reflect only changes post-baseline. Results are presented for
the AUC measure adjusted for baseline cortisol as the latter provides a more con-
servative estimate of the actual cortisol “response” to the challenge. Results were
unchanged with analyses using the measure of AUC that included baseline cortisol.
Repeated measure ANOVA analyses were also examined to test for gender-by-age
differences in patterns of cortisol response to the challenge session. For the repeated
measures analyses, Greenhouse–Geisser non-sphericity corrections were applied, as
recommended (Vasey and Thayer, 1987). Uncorrected degrees of freedom are
reported along with a non-sphericity correction factor (epsilon) and the adjusted P-
value. SAS 6.12 software was used for all analyses.

3. Results
Twenty-six younger subjects, 9 men and 17 women, ages 22–36 (x̄=26.1, SD=3.8),
and 14 older adults, 7 men and 7 women, ages 67–88 (x̄=77.5, SD=6.1) participated
in the study. Due to the strict selection criteria, there were no group differences on
possible correlates of cortisol activity such as medication use or relative weight (body
mass index), and no pre-existing health conditions such as diabetes or CVD in either
group. There were no differences in educational attainment (x̄=16 years in both
younger and older subjects; P=0.44) so education was not considered further as a
covariate. Likewise, analyses of possible differences in cortisol activity by OC status
did not reveal any differences so OC-use was not considered further as a covariate.
The younger subjects were marginally more likely to be classified as being depressed
(31% [n=8] of the young subjects vs. 7.1% [n=1] of the older subjects; χ2=2.91,
P=0.09). Those classified as depressed exhibited higher baseline cortisol (x̄=0.13
ug/dl vs. x̄=0.09 ug/dl among those classified as non-depressed, t=2.17, P=0.04) and
less relative increases in response to the challenge (x̄=1.31 ug/dl vs. x̄=2.03 ug/dl
among those classified as non-depressed, t=2.38, P=0.02 for maximum increase).
Depression was included as a control variable in subsequent analyses. All analyses
were also examined with those scoring 16+ on the CESD excluded from the analyses;
all results reported here were unchanged from the analyses including all subjects
and controlling for depression.
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 231

For baseline salivary cortisol, there was a significant main effect for age
[F(1,33)=19.14, P=0.0001; with significantly higher levels seen among the older sub-
jects (x̄=0.20 µg/dl among older subjects vs. x̄=0.11 µg/dl among the younger sub-
jects, P⬍0.001). There was also a marginal age-by-gender interaction [F(1,33)=3.79,
P=0.06], suggesting that this relationship deserves further exploration with larger
data sets. As shown in Fig. 1, there was no significant gender difference among the
younger subjects (P=0.99). However among the older subjects, the men had signifi-
cantly higher baseline cortisols (x̄=0.23 µg/dl vs. 0.16 µg/dl among the women,
P=0.02).
With respect to cortisol reactivity to the cognitive challenge, a significant age-by-
gender difference was seen for maximum increase over baseline [F(1,32)=11.70;
P=0.002]. As shown in Fig. 2, planned comparisons for this interaction revealed that
among the younger subjects, men exhibited a greater relative percentage increase
over baseline (i.e., maximum increase/baseline) as compared with the women
(x̄=264% for men vs. 112% for women, P=0.004). The reverse pattern was seen in
the older group where older women exhibited greater relative increase compared
with older men (x̄=228% for women vs. 88% for men, P=0.05).
Analyses of total AUC adjusted for baseline revealed a similar pattern, though
the age-by-gender interaction achieves only marginal statistical significance
[F(1,33)=3.52, P=0.07]. As shown in Fig. 3, younger men had greater overall

Fig. 1. Comparisons of baseline salivary cortisol levels across age and gender groupings (group differ-
ences are indicated by paired symbols; ***P⬍0.001; +P⬍0.05; 苲P=0.10).
232 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

Fig. 2. Comparisons of maximum increase in cortisol during the challenge session across age and gender
groupings (group differences are indicated by paired symbols: *P⬍0.005; 苲P⬍0.05).

response (17.5 vs. 7.8 for young women, P=0.05) whereas older women tended to
show greater overall response compared with older men, though the differences in
this case were not statistical significance (21.7 for the women vs. 16.2 for the men,
P=0.41). There was also a non-significant trend towards greater AUC among older
adults (18.9 AUC among older adults vs. 12.6 for the younger group, P=0.14). Analy-
ses of the unadjusted AUC data (i.e., total cortisol exposure including baseline levels
plus challenge responses) revealed a significant age effect [F(1,33)=7.29, P=0.01]
with older adults showing a total AUC 64% larger than that seen among the younger
subjects: 35.8 vs. 21.9. This latter finding is consistent with the previous findings of
higher baseline cortisols among the older adults as well as marginally greater adjusted
AUCs among the older adults.
Actual patterns of response were examined initially through repeated measures
ANOVA. A significant time effect was found [F(8,232)=5.09, P=0.015, e=0.20]
reflecting the fact that our challenge protocol successfully elicited increases in cor-
tisol activity. A significant time*sex*age interaction was also found [F(8,232)=5.14;
P=0.01, e=0.20]. The time trends in cortisol response underlying this interaction were
further examined by plotting cortisol responses over the seven time-points beyond
the baseline, defined as deviations from the baseline level for each individual. Fig.
4 shows the mean responses by gender and category of trajectory — i.e. responder
or non-responder — for the young and old groups separately. Simultaneous 90%
confidence intervals are shown for all groups (Miller, 1981). Most notable is the fact
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 233

Fig. 3. Comparisons of total area under the response curve across age and gender groupings (group
difference are indicated by paired symbols: *P=0.05; @P=0.02).

that among the younger subjects, it is the men “responders” who exhibit the largest
responses while among the older subjects, it is the women “responders” who exhibit
the larger responses. For both age groups, the high responders — young men and
older women — lie on trajectories that are separated from the non-responder tra-
jectories from time 15 c (i.e. first challenge sample) through to time 120 r (i.e., 120
minutes after the end of challenge and the end of recovery period). The young female
responders are significantly above both non responder groups from time 15 c through
to time 60 r (i.e., 60 minutes into the recovery period). The older male responders
are significantly above the non-responders only at times 30 r and 60 r (i.e., 30 and
60 minutes respectively into the recovery period after the conclusion of the
challenge). The individual trajectories of these men show a time delay in response
to challenge relative to the rapid high cortisol response of the older women who are
classified as responders.
Examination of the age-by-gender-specific distributions of these patterns of
response vs. non-response to the challenge indicates that among the younger subjects,
the men were more likely to be responders (3/4 men versus 1/9 women; c2=5.3,
P=0.02). By contrast, among the older subjects, the men and women are more compa-
rable in their propensity to show a response to the challenge (2/6 men and 2/5
women; c2=0.052, P=0.82). As illustrated in Fig. 4, however, the female responders
among these older subjects exhibit considerably larger elevations in response to the
challenge than do the male responders.
234 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

Fig. 4. (A) Salivary cortisol response to the cognitive challenge by age and gender groups: deviations
from baseline among younger men and women. (B) Salivary cortisol response to the cognitive challenge
by age and gender groups: deviations from baseline among older men and women.

4. Discussion

Analyses of data on salivary “free” cortisol responses to a standardized cognitive

challenge in younger and older men and women revealed the hypothesized age-by-
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 235

gender interaction. For both maximum increase in cortisol as well as overall total
area under the response curve, younger men were found to exhibit greater cortisol
response to the challenge as compared with younger women while the reverse was
true in the older adults (i.e., older women were found to exhibit greater response as
compared with older men). Main effects for age were also seen for baseline cortisol
and total AUC. In each case, the older adults had significantly higher cortisol values
(i.e., higher baseline cortisol levels and greater total AUC).
These findings are consistent with previous studies that have suggested greater
age-related increase in 24-hr, unstimulated cortisol activity in women. Using pooled
data from a number of studies of circadian cortisol rhythms, van Cauter et al. (1996)
found that postmenopausal women exhibited shorter nocturnal nadirs, higher morning
acrophase and higher overall mean 24-hr cortisols as compared with younger women
and younger and older men. As indicated earlier, a growing body of data also sug-
gests that women experience a greater age-related increase in HPA axis response to
challenge, though none of these studies has directly compared responses of younger
and older men and women to a single specific challenge. At a younger age, cortisol
response to psychological challenge has more frequently been found to be greater
among men (Collins and Frankenhaeuser, 1978; Frankenhaeuser et al., 1978; Kirsch-
baum et al., 1992b) though one study (Stoney et al., 1987) found no significant
gender difference. Responses to pharmacological challenge have generally not shown
consistent gender differences in patterns of response among younger adults (Gallucci
et al., 1993; Streeten et al., 1984). Studies of older men and postmenopausal women,
by contrast, have shown an intriguing pattern of greater HPA axis reactivity among
women in response to pharmacologic (Greenspan et al., 1993; Heuser et al., 1994;
Peskind et al., 1995; Wilkinson et al., 1997) and non-pharmacologic challenge
(Seeman et al., 1995). The data from our current study represent the first direct test
(and confirmation) of the apparent age-by-gender interaction with respect to HPA
axis reactivity.
The apparent greater age-related shift towards increased HPA axis responsivity to
challenge in women is intriguing. The possible role of reproductive hormones such as
estrogen in this apparent age-related shift towards greater relative HPA axis reactivity
among postmenopausal women is suggested by several lines of evidence. First, vari-
ations in estrogen levels have been linked to altered reactivity — periods of higher
estrogen being associated with reduced blood pressure (von Eiff et al., 1971), cardio-
vascular (Sita and Miller, 1996) and HPA reactivity (Bonen et al., 1991; Marinari
et al., 1976). Second, estrogen replacement therapy has also been related to
reductions in blood pressure responses to challenge (Stoney, 1992) and a blunting
of cortisol responses to cognitive and physical stress tests in postmenopausal women
exposed to transdermal application of 17β-estradiol for six weeks (Lindheim et al.
1992, 1994). Less clear, however, is the potential impact of most common forms of
hormone-replacement therapy (HRT) (i.e., the combined equine estrogens in Prema-
rin and frequent concomitant use with a progestogen) on HPA axis reactivity. A
study by Burleson et al. (1998) reports that postmenopausal women on HRT exhibit
higher plasma cortisol levels and show higher levels of cortisol in response to chal-
lenge. However, as these authors point out, the higher plasma cortisol levels seen
236 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240

in their women on HRT likely reflect the fact that exogenous estrogen simulates
increased production of cortisol-binding globulin (CBG), leading to higher levels of
bound cortisol. Notably, levels of unbound or free (biologically active) cortisol levels
have been reported to remain essentially unchanged (Sapolsky, 1992). Thus, the
reported finding of higher plasma cortisol levels in the women on HRT leaves
unanswered the question of whether most commonly prescribed forms of HRT (i.e.,
Premarin with or without progestogen) influence levels of free (biologically active)
cortisol. Interestingly, recent data from a pilot study using measures of free cortisol
(based on salivary cortisol) suggest that use of Premarin alone (i.e., unopposed by
progesterone) is associated with attenuated reactivity to challenge while use of com-
bined HRT is associated with less attenuation of reactivity (Seeman, unpublished
data).
Effects of estrogen on HPA axis reactivity have been hypothesized to operate at
multiple levels including pituitary responsiveness to CRH and suprapituitary levels
(Fujimoto et al., 1986; Jacobs et al., 1989; Young, 1995). At suprapituiatary levels,
the plausibility of estrogenic effects on HPA axis regulation is suggested by evidence
of oestradiol-receptors in the hippocampus and hypothalamus (Teyler et al., 1986),
both brain regions involved in HPA axis regulation. Moreover, animal studies have
demonstrated several important effects of estrogen on brain mechanisms relevant to
HPA axis activity such as regulation of synaptogenesis in the CA1 region of the
hippocampus (McEwen and Woolley, 1994) and induction of rapid changes in elec-
trical activity and membrane properties of both hippocampal (Foy et al., 1982; Wong
and Moss, 1991) and hypothalamic (Minami et al., 1990) neurons. Research has also
demonstrated that 17β-estradiol exposure induces filopodial growth in hippocampal
neurons within minutes, a time base consistent with 17β-estradiol-related enhance-
ment of neuronal cell excitability (Brinton, 1993), possibly through direct action on
neuronal membrane properties (Wong et al., 1996). These data provide important
evidence that there is continued malleability of the brain, and specifically hippocam-
pal synaptic plasticity in response to estrogen in adult animals. Estrogen may also
counteract some of the deleterious effects of glucocorticoids on neurons, having been
shown to augment cerebral glucose utilization (Bishop and Simpkins, 1995) and to
have antioxidant properties that may help blunt neurotoxic effects of free radicals
(Mooradian, 1993; Niki and Nakano, 1990). Thus, the presence of estrogen may
contribute to reduced cortisol reactivity through mechanisms that protect and promote
negative feedback within the HPA axis.
At the level of the pituitary and adrenals, there appears to be greater age-related
increase in ACTH response to CRH/VP stimulation in women (Born et al., 1995)
and greater pituitary resistance to negative feedback from cortisol (Wilkinson et al.,
1997); both possibly related to the age-related changes in estrogen levels in women.
In combination with women’s apparent greater adrenal responsiveness to ACTH
(Born et al., 1995; Horrocks et al., 1990; Roelfsema et al., 1993), these age-related
changes would result in considerably enhanced cortisol response to challenge. In
light of these various data, it appears reasonable to suggest that the presence of
estrogen may contribute to reduced cortisol reactivity through mechanisms that pro-
tect and promote negative feedback to the HPA axis at multiple levels.
 T.E. Seeman et al. / Psychoneuroendocrinology 26 (2001) 225–240 237

Further research is needed to replicate the findings reported here and to extend
our understanding of the underlying mechanisms for any gender difference in age-
related trends towards increased HPA axis activity (basally and/or in response to
challenge). If confirmed, this increased HPA axis reactivity to challenge at an older
age in women may represent one underlying mechanism for the relative decline
postmenopause in women’s protection against major sources of morbidity and mor-
tality such as cardiovascular disease, osteoporosis and cancer. Further, if HRT is
found to attenuate HPA axis reactivity, this could represent an additional benefit of
HRT with respect to health and functioning at older ages.

Acknowledgements

This research was supported by NIA grants AG–00586 and AG–17056 to TES,
by the MacArthur Research Network on Successful Aging through a grant from the
John D. and Catherine T. MacArthur Foundation, and by NIA grants AG–10418 to
that UCLA Older Americans Independence Center, AG–10469 to the Yale University
Claude Pepper Older Adults Center and AG–05142 to the USC Alzheimer’s Disease
Research Center. The authors would like to express their appreciation and thanks to
USC–ADRC Clinical Coordination Center for providing access to their pool of heal-
thy older volunteers; to the volunteers themselves for their participation and assist-
ance with this research project; to Kate Leung, Deborah Buckwalter and Doug Cox
who each made invaluable contributions to the successful implementation of this
project; and to Elizabeth Colasurdo and Richard Vertz for performing the salivary
cortisol assays.

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