Psychoneuroendocrinology (2006) 31, 642–652

www.elsevier.com/locate/psyneuen

Gender differences in hypothalamic–pituitary–

adrenal (HPA) axis reactivity
Magdalena Uharta, Rachel Y. Chonga, Lynn Oswaldb,
Ping-I Linc, Gary S. Wanda,b,*
a
Department of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue,
Room 863, Baltimore, MD 21205, USA
b
Department of Psychiatry, The Johns Hopkins University School of Medicine, 550 North Broadway,
Suite 506, Baltimore, MD 21205, USA
c
Section of Medical Genetics, Department of Medicine, Center for Human Genetics, Duke University,
595 LaSalle Street, Durham, NC 27710, USA

Received 5 May 2005; received in revised form 12 December 2005; accepted 8 February 2006

KEYWORDS Summary The present study was designed to determine whether there are
Gender; gender differences in hormonal response patterns to HPA axis activation. To this end,
Cortisol; two methods of activating the HPA axis were employed: a standardized psychological
Adrenocorticotropin stress test and a pharmacological challenge.
(ACTH); Healthy subjects (mean age 23.4 years, SD 7.0 years) completed a naloxone
Hypothalamic–pitu- challenge and/or the modified Trier Social Stress Test (TSST). For the naloxone
itary–adrenal (HPA) challenge, two baseline blood samples were obtained. Placebo was then
axis; administered (0 min), followed by increasing doses of intravenous naloxone (50,
Trier social stress test; 100, 200 and 400 mg/kg) at 30-min intervals. Post-placebo blood samples were
Naloxone challenge collected at 15-min intervals for 180 min. The TSST consisted of 5 min of public
speaking followed by 5 min of mental arithmetic exercises. Three baseline and five
post-TSST blood samples were drawn.
Eighty subjects (53 male, 27 female) underwent the TSST. Following the
psychological stressor, adrenocorticotropin (ACTH) and cortisol responses were
significantly greater in male subjects compared to female subjects (zZK2.34, pZ
0.019 and zZK2.12, pZ0.034, respectively). Seventy-two subjects (52 male, 20
female) underwent HPA axis activation induced by naloxone. In contrast to the TSST,
cortisol responses to the naloxone challenge were significantly greater in female
subjects compared to male subjects (zZ4.11, p!0.001). Forty-one subjects (25
male, 16 female) completed both the TSST and naloxone challenge. In this subset,
ACTH and cortisol responses to the TSST did not differ significantly by gender,
although the effect size was moderate to large. Adrenocorticotropin and cortisol

* Corresponding author. Address: Division of Endocrinology, The Johns Hopkins University School of Medicine, Ross Research Building,
Room 863, 720 Rutland Avenue, Baltimore, MD 21205, USA. Tel.: C1 410 955 7225; fax: C1 410 955 0841.
E-mail address: gwand@jhmi.edu (G.S. Wand).

0306-4530/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2006.02.003
Gender and HPA axis hormonal response 643

responses to the naloxone challenge were significantly greater in female subjects

compared to male subjects (zZ2.29, pZ0.022 and zZ4.34, p!0.001, respectively).
In summary, male subjects had greater HPA axis responses to a psychological
stressor than female subjects, and females had greater hormonal reactivity than
males to pharmacological stimulation with naloxone. Such differences are of interest
as potential contributors to gender differences in health risks.
Q 2006 Elsevier Ltd. All rights reserved.

1. Introduction In response to psychological stressors in young

Activation of the hypothalamic–pituitary–adrenal
(HPA) axis is an essential adaptive mechanism that
enables the human body to maintain physiological
stability in response to stressful stimuli (Herman
and Cullinan, 1997; Chrousos 1998; Tsigos and
Chrousos, 2002). Following the perception of stress,
corticotropin-releasing hormone (CRH) neurons in
the hypothalamus receive regulatory impulses from
several major neurotransmitter systems, including
direct and indirect inhibitory signals from
b-endorphin-producing neurons (Jackson et al.,
1990; Calogero, 1995; Jessop, 1999). CRH release
stimulates the synthesis and release of adreno-
corticotropin (ACTH) by the anterior pituitary,
which in turn stimulates the synthesis and release
of cortisol by the adrenal cortex. There is evidence
that healthy individuals react differently to stress-
ful stimuli (Berger et al., 1987; Kirschbaum et al.,
1995a), and that both enhanced and attenuated
hormonal responses to stress are maladaptive.
Chronic HPA axis dysregulation is associated with
the development of mood and anxiety disorders,
such as depression (Sapolsky, 2000; Gold and
Chrousos, 2002; Sherwood Brown et al., 2004).
Moreover, excess cortisol exposure is related to a
variety of medical conditions including hyperten-
sion, atherosclerosis, obesity, insulin resistance,
dyslipidemia, bone demineralization, and impaired
immunity (McEwen, 1998; Tsigos and Chrousos,
2002). Likewise, it contributes to the development
and maintenance of substance use disorders, such
as alcohol dependence (Gianoulakis, 1998). Inter-
estingly, pronounced differences in the prevalence
of several of these disorders have been shown
between men and women (Boyd and Weissman,
1981; Grant et al., 2004).
Previous studies suggest that gender influences
the HPA axis hormonal responses to stress. In
preclinical models, ACTH and corticosterone levels
in response to stress have been shown to be
consistently greater in females compared with
males (Kitay, 1961; Handa et al., 1994; Armario
et al., 1995). However, in human studies, no such
clear-cut gender differences have been established.
subjects, certain studies have shown higher cortisol
and ACTH responses in male subjects compared to
females (Kirschbaum et al., 1992, 1995a,b). Never-
theless, other studies have suggested that there are
no significant gender differences in young subjects
in response to stress (Collins and Frankenhaeuser,
1978; Frankenhaeuser et al., 1978). In a study of
healthy young adults, Kirschbaum et al. (1999)
showed that ACTH responses were elevated in men
compared to women and that free cortisol
responses were similar between men and women
in the luteal phase of their menstrual cycle whereas
women in the follicular phase or taking oral
contraceptives showed lower free cortisol
responses compared to males. The same gender
effect was demonstrated in elderly subjects, with
higher ACTH and cortisol responses in male subjects
compared to females (Kudielka et al., 1998;
Traustadottir et al., 2003). Conversely, higher
HPA axis hormonal responses to stress in elderly
females compared to males have also been
reported (Seeman et al., 1995, 2001). Thus, the
impact of gender on the HPA axis hormonal
response to stressful events remains inconclusive.
Activation of the HPA axis can be evoked by
numerous methods that act at different levels of
the HPA system. Removing the endogenous inhibi-
tory opioid tone on CRH neurons using naloxone, a
non-selective opioid receptor antagonist, induces a
rise in ACTH and cortisol (Volavka et al., 1979;
Morley et al., 1980; Wand et al., 1998) and thus
provides an assessment strategy for the functional
evaluation of the hypothalamic opioid tone.
Although several studies have evaluated effects of
opioid blockade on the HPA axis (Cohen et al., 1983;
Kreek, 1996; Wand et al., 1999, 2001), little
research has addressed gender differences in the
opioid–HPA axis interactions in healthy subjects.
The aim of the present study was to determine
whether there are gender differences in HPA axis
hormonal response patterns to two different
methods of HPA axis activation: a standardized
psychological stress test and a pharmacological
challenge with naloxone.
644
2. Methods
2.1. Subjects
One hundred and eleven healthy subjects between
the ages of 18 and 50 (mean age 23.4 years, SD 7.0
years) from the Baltimore area were recruited by
newspaper advertisements and posted fliers. Per-
sons who appeared to qualify for research partici-
pation based on a telephone screen were invited to
the laboratory for an interview. After being given
complete description of the study, volunteers
provided written informed consent for the protocol
approved by the Johns Hopkins Medicine Insti-
tutional Review Board. Subject assessment
included a medical history and physical exam
performed by a physician, a complete blood
count, comprehensive metabolic panel (including
renal and hepatic function tests), electrocardio-
gram, urinalysis, alcohol breathalyzer test and
urine toxicology screen. A urine pregnancy test
was obtained on female subjects. The semi-
structured assessment for the genetics of alcohol-
ism (SSAGA) (Bucholz et al., 1994) was administered
by a master’s degree-level interviewer to identify
DSM-IV axis I psychiatric diagnoses.
Exclusion criteria were as follows: (a) presence
of a serious medical condition, (b) presence of a
DSM-IV axis I disorder, including alcohol/drug abuse
or dependence, (c) use of any psychoactive
medications within the past 30 days, (d) treatment
in the last 6 months with any medication that may
affect opioid or HPA axis function, including
antidepressants, neuroleptics, sedative hypnotics,
glucocorticoids, appetite suppressants, estrogens
(including anti-contraceptive pills), opiates, or
dopamine medications, (e) presence of a seizure
disorder or history of closed head trauma, (f)
consumption of more than 30 alcoholic drinks per
Table 1 Demographic characteristics by gender.
TSST, nZ80a
Male
Sample size 53
Age, mean (SD), (years) 26.7 (10.1)
Race, no. (%)
Caucasian 40 (75.5)
African–American 8 (15.1)
Asian 5 (9.4)
BMI, mean (SD), (kg/m2) 25.2 (3.5)
Estradiol, mean (SE), (pg/mL)
Progesterone, mean (SE), (ng/mL)
a
Eighty subjects underwent the TSST and 72 subjects completed the naloxone challenge. Of these, 41 subjects completed both
sessions. Thus, there were 111 subjects in all.
M. Uhart et al.
month, (g) positive urine toxicology screen, (h) or,
for females, pregnancy or lack of effective non-
hormonal methods of birth control. Menstrual cycle
phase was determined by measurements of estra-
diol and progesterone levels on the day of the
challenge (Table 1). Subjects with progesterone
levels greater than or equal to 3 ng/mL were
categorized as being in the luteal phase of their
menstrual cycle (Yen et al., 1999).
Eighty subjects underwent the TSST and 72
subjects completed the naloxone challenge. Of
these, 41 subjects completed both sessions. Thus,
there were 111 subjects in all. In the TSST group,
there were 53 male subjects and 27 female
subjects. Based on progesterone levels, all female
subjects completed the TSST during the follicular
phase of their menstrual cycle. In the naloxone
group, there were 52 male subjects and 20 female
subjects. Based on progesterone values, all female
subjects underwent the naloxone challenge during
the follicular phase of their menstrual cycle.
2.2. General procedure
Following the initial assessment interview, subjects
reported to the Johns Hopkins Hospital Outpatient
General Clinical Research Center (GCRC) to com-
plete either the naloxone challenge and/or the
Trier Social Stress Test (TSST). For the subset of
subjects who completed both challenges, one
challenge was completed on one day, and the
other challenge on a separate day. The time
between the administrations of both challenges
was 7–14 days, and the order in which the
challenges were completed was randomized. Sub-
jects were instructed to get adequate sleep the
night prior to the challenges and to report any
stressful situations that may have occurred during
the previous week. They were also instructed to
Naloxone challenge, nZ72a
Female Male Female
27 52 20
22.6 (5.3) 21.4 (2.5) 20.9 (2.6)
17 (63.0) 43 (82.7) 12 (60.0)
10 (37.0) 5 (9.6) 7 (35.0)
0 (0.0) 4 (7.7) 1 (5.0)
23.9 (3.9) 24.3 (3.0) 24.9 (3.5)
46.3 (8.0) 49.7 (14.1)
0.5 (0.1) 0.7 (0.1)
Gender and HPA axis hormonal response
refrain from any alcohol, illicit drugs, or over-the-
counter medications for 48 h prior to participating
in the study protocol. Urine toxicology screens were
completed before each session. On the day of each
challenge, subjects fasted from 1000 h until testing
was completed.
2.3. Trier Social Stress Test (TSST)
The TSST consists of 5 min of public speaking
followed by 5 min of a mental arithmetic task and
was completed as previously described in detail
(Kirschbaum et al., 1993; Uhart et al., 2004). Upon
arrival at the GCRC, an intravenous catheter was
inserted into a forearm vein at 1200 h. Baseline
blood samples for cortisol and ACTH were obtained
at 1300, 1315, and 1330 h. Subjects then partici-
pated in the TSST. Immediately following com-
pletion of the TSST and at 15-min intervals, five
additional blood specimens were drawn for ACTH
and cortisol.
2.4. Naloxone challenge
For the naloxone challenge, subjects received five
doses of naloxone according to the 1 day, single-
session protocol reported by Mangold et al.
(2000). Upon arrival at the GCRC, subjects had
an intravenous catheter inserted into a forearm
vein at 1200 h. One hour later, a bolus of 0.9%
saline was administered as a placebo; this was
designated as time 0 min. Subsequently, incre-
mental doses of naloxone dissolved in 0.9% saline
were administered at 30, 60, 90, and 120 min. In
all, a total of five increasing doses of naloxone
were administered (0, 50, 100, 200 and 400 mg/
kg). Baseline blood samples for cortisol and ACTH
were obtained 15 min before and immediately
prior to placebo administration. Post-placebo
blood samples for cortisol and ACTH were drawn
at 15-min intervals for 180 min.
2.5. Hormone assays
Hormones were assayed as previously described
(Blevins et al., 1994). Plasma concentrations of
ACTH were assayed by a two-site IRMA (Nichols
immunoradiometric assay). Plasma concentrations
of cortisol were measured by radioimmunoassay
(Diagnostic Products Corporation, Inc., Los
Angeles, CA, USA). Plasma concentrations of
estradiol and progesterone were measured in
female subjects (Diagnostic Products Corporation,
Inc., Los Angeles, CA, USA). Intra-assay and inter-
645
assay coefficients of variance for all assays was less
than 10%.
2.6. Statistical analysis
Preliminary analyses included evaluation of gender
group differences in demographic characteristics
with t-tests for continuous variables (age and body
mass index) and c 2 analyses for categorical
variables (race). The major outcomes of interest
were hormonal measurements of ACTH and corti-
sol. All hormonal measurements were transformed
to the logarithmic scale due to non-normality. The
mean baseline for each variable was calculated by
taking the average of the baseline measurements
(K30, K15, and 0 min time points for the pre-TSST
measurements and K15 and 0 min time points for
the pre-naloxone challenge measurements). The
mean baseline differences in hormonal levels
between gender groups were also analyzed with
t-tests.
We then carried out four different sets of
analyses to assess the effect of gender on
hormone responses in each of the two challenge
groups, and in the subset of subjects who
completed both challenges. For each analysis,
age and race were incorporated into the model
for the TSST data whereas age, race, baseline
hormones and BMI were incorporated into the
model for the naloxone data set. Baseline
hormone values were added because baseline
hormone values differed by gender for the
naloxone challenge group only; BMI was added
because a pharmacological agent was adminis-
tered. First, we performed longitudinal data
analysis. Each hormone measurement at each
time point was treated as the outcome in the
generalized linear model using generalized esti-
mating equations (GEE) to take into account the
within-individual correlation residuals arising from
repeated measurements for each individual (Zeger
and Liang, 1986). The model included a contrast
for gender difference as a major covariate of
interest, time, and time squared to adjust for
non-linear time trend. Second, we conducted
post-hoc regression analyses to evaluate gender
effect on hormone differences at each time point.
Third, we compared gender differences in peak
hormone responses to the TSST and naloxone
challenge. Peak hormone level was defined as the
highest level reached during each of the chal-
lenges. We treated the peak hormone response as
the outcome variable and gender as the major
covariate of interest in the generalized linear
model. Fourth, we carried out area under the
646 M. Uhart et al.

generalized linear model. Lastly, we calculated

the effect size of the hormonal responses in each
of the two challenge groups, and in the subset of
subjects who completed both challenges. Effect
size was calculated as the difference between the
mean peak hormone response values of the male
group and female group divided by the standard
deviation of the overall sample (Cohen, 1988).
The analyses were two sided with a 0.05
significance level and were performed by using
the software STATA 8.0. Finally, we plotted the
unadjusted means of ACTH and cortisol response
concentrations to the two challenges against time
by gender.

3. Results

3.1. Demographics

Demographic characteristics for the subjects

Figure 1 (a) Plasma ACTH response to TSST by gender.
Values reflect unadjusted means (SE). *Plasma ACTH
levels differed significantly by gender at the following
time points: 25 min, pZ0.007; 40 min, pZ0.021. (b)
Plasma cortisol response to TSST by gender. Values
reflect unadjusted means (SE). *Plasma cortisol levels
differed significantly by gender at the following time
points: 25 min, pZ0.027; 85 min, pZ0.006.
undergoing the TSST (nZ80) and the naloxone
challenge (nZ72) are in Table 1. Subjects were
healthy and predominantly Caucasian, and all
subjects were non-smokers. The groups differed in
racial composition (TSST, c2Z6.76, pZ0.034;
naloxone, c2Z6.70, pZ0.035), and race was
adjusted for in our statistical models. There were
no statistically significant differences between
gender groups in terms of age and BMI. In the
TSST group, males were 18–50 years (mean 26.7
years, SD 10.1 years) and females were 18–42 years
(mean 22.6 years, SD 5.3 years). In the naloxone
group, males were 18–32 years (mean 21.4 years, SD
2.5 years) and females were 18–27 years (mean 20.9
years, SD 2.6 years).

curve (AUC) analysis. ACTH and cortisol AUC 3.2. Trier social stress test (TSST)
values by gender were computed by using the
trapezoid algorithm and the effect of gender on There were no mean baseline differences in plasma
differences in the AUC was assessed by using the ACTH and cortisol levels by gender (tZ1.49, pZ

Table 2 Adjusted mean hormonal values for the TSST and naloxone challenge by gender.
Hormone/measure TSST, nZ80 Naloxone challenge, nZ72
Male Female p Value Male Female p Value
Sample size 53 27 52 20
ACTH
AUC, mean (SE) 232.2 (7.5) 209.2 (8.7) zZK1.93, pZ0.054 517.1(8.5) 546.4 (15.0) zZ1.63, pZ0.103
Peak, mean (SE) 29.1 (1.1) 19. 5 (1.1) zZK2.42, pZ0.016 32.1(1.0) 44.7(1.1) zZ2.68, pZ0.007
Cortisol
AUC, mean (SE) 201.2 (5.4) 185.7 (7.9) zZK1.59, pZ0.111 458.8 (5.5) 500.5 (8.7) zZ3.99, p!0.001
Peak, mean (SE) 17.9 (1.0) 13.7 (1.1) zZK2.15, pZ0.031 20.4(1.0) 27.9(1.0) zZ5.06, p!0.001
ACTH, adrenocorticotropic hormone; AUC, area under the curve.
Gender and HPA axis hormonal response 647

0.139 and tZ1.70, pZ0.092, respectively). 3.3. Naloxone challenge

Following the psychological stressor, ACTH and
cortisol responses to the TSST were significantly
greater in male subjects compared to female
subjects (zZK2.34, pZ0.019 and zZK2.12, pZ
0.034, respectively) (Fig. 1a and b). Male subjects
also had greater peak ACTH and cortisol responses
compared to female subjects (zZK2.42, pZ0.016
and zZK2.15, pZ0.031, respectively). In addition,
ACTH area under the curve responses were margin-
ally greater in males as compared to females
(zZK1.93, pZ0.054) (Table 2).
There were no mean baseline differences in plasma
cortisol levels by gender (tZ1.09, pZ0.281).
However, ACTH baseline levels differed by gender
(tZ2.57, pZ0.012) and baseline hormonal levels
were used as a covariate in the analysis. Cortisol
responses to the naloxone challenge were signifi-
cantly greater in female subjects compared to male
subjects (zZ4.11, p!0.001). In addition, ACTH
responses were marginally greater in female
subjects compared to male subjects (zZ1.70,
pZ0.089) (Fig. 2a and b). Female subjects also
had statistically significantly greater peak ACTH and
cortisol responses compared to male subjects (zZ
2.68, pZ0.007 and zZ5.06, p!0.001, respect-
ively). In addition, female subjects had greater
cortisol area under the curve response compared to
males (zZ3.99, p!0.001) (Table 2).

3.4. Trier Social Stress Test (TSST) and

naloxone challenge

Forty-one subjects completed both the TSST and

the naloxone challenge. Demographic character-
istics are presented in Table 3. There were no
statistically significant differences between gender
groups in terms of age, race and BMI.
Adrenocorticotropin and cortisol responses to
the TSST did not differ significantly by gender
(Fig. 3a and b, and Table 4). Given the small sample
size and lack of significance, effect sizes were
calculated showing Cohen’s dZ0.58 and 0.77 for
ACTH and cortisol, respectively.
However, ACTH and cortisol responses to the
naloxone challenge were significantly greater in

Table 3 Demographic characteristics by gender in

subjects who completed both the TSST and naloxone
challenge.
TSST and naloxone challenge, nZ41
Male Female
Figure 2 (a) Plasma ACTH response to naloxone by Sample size 25 16
gender. Values reflect unadjusted means (SE). Pl denotes Age, mean (SD), (y) 20.8 (2.8) 20.5 (2.4)
time of placebo (saline) administration. N denotes times Race, no. (%)
of incremental naloxone administration. (b) Plasma Caucasian 20 (80) 11 (68.7)
African–American 2 (8) 5 (31.3)
cortisol response to naloxone by gender. Values reflect
Asian 3 (12) 0 (0)
unadjusted means (SE). Pl denotes time of placebo
BMI, mean (SD), 24.8 (3.3) 25.1 (3.6)
(saline) administration. N denotes times of incremental (kg/m2)
naloxone administration. *Plasma cortisol levels differed TSST Naloxone
significantly by gender at the following time points: Estradiol, mean (SE), 48.8 (11.1) 46.1 (16.6)
60 min, pZ0.050; 75 min, pZ0.009; 90 min, p!0.001; (pg/mL)
105 min, p!0.001; 120 min, p!0.001; 135 min, p! Progesterone, mean 0.5 (0.1) 0.7 (0.1)
0.001; 150 min, p!0.001; 165 min, p!0.001; 180 min, (SE), (ng/mL)
p!0.001.
648 M. Uhart et al.

cortisol responses compared to male subjects (zZ

3.24, p!0.001 and zZ5.0, p!0.001, respectively).
In addition, female subjects had greater ACTH and
cortisol area under the curve response compared to
males (zZ2.30, pZ0.022 and zZ4.25, p!0.001)
(Table 4).

4. Discussion

In the present study, we observed that healthy male

Figure 3 (a) Plasma ACTH response to TSST by gender
in subjects who completed both the TSST and naloxone
challenge. Values reflect unadjusted means (SE). (b)
Plasma cortisol response to TSST by gender in subjects
who completed both the TSST and naloxone challenge.
Values reflect unadjusted means (SE).
female subjects compared to male subjects (zZ
2.29, pZ0.022 and zZ4.34, p!0.001, respect-
ively) (Fig. 4a and b). Female subjects also had
statistically significantly greater peak ACTH and
subjects demonstrate a more robust ACTH and
cortisol response to a psychological stress
compared to females. In contrast, healthy females
had a higher cortisol and marginally higher ACTH
response to naloxone compared to males. Overall,
our findings were consistent whether our analysis
was conducted using a between-subject or within-
subject design.
Our findings in response to the TSST lend further
support to a number of studies that have shown
greater cortisol and ACTH responses in young and
elderly male subjects to a psychological challenge,
including public speaking and mental arithmetic
exercises, compared to females (Kirschbaum et al.,
1992, 1995a,b; Kudielka et al., 1998). The evidence
in human studies of the effect of gender on HPA axis
stress response, however, has been conflicting.
Certain studies have suggested higher HPA axis
responses in elderly females compared to elderly
males employing a driving simulation and a
cognitive paradigm challenge (Seeman et al.,
1995, 2001), and following lumbar puncture (Petrie
et al., 1999). Still, other studies have reported no
significant gender difference in cortisol response to
examination stress and a cognitive-conflict task in
young subjects (Collins and Frankenhaeuser, 1978;
Frankenhaeuser et al., 1978).
Interestingly, in contrast to the findings after
exposure to psychological stress, we observed

Table 4 Adjusted mean hormonal values for the TSST and naloxone challenge by gender in subjects who completed
both the TSST and naloxone challenge.
Hormone/ TSST, nZ41 Naloxone challenge, nZ41
measure
Male Female p Value Male Female p Value
Sample size 25 16 25 16
ACTH
AUC, mean (SE) 221.9 (9.7) 205.0 (11.3) zZK1.07, pZ0.284 512.3 (14.2) 563.9 (17.3) zZ2.30, pZ0.022
Peak, mean (SE) 27.9 (0.1) 19.8 (1.1) zZK1.53, pZ0.125 31.1 (1.0) 47.9 (3.0) zZ3.24, p!0.001
Cortisol
AUC, mean (SE) 197.3 (7.7) 181.6 (11.5) zZK1.08, pZ0.281 466.5 (9.3) 522.2 (9.1) zZ4.25, p!0.001
Peak, mean (SE) 17.6 (1.0) 13.4 (1.1) zZK1.54, pZ0.124 22.2 (1.0) 30.8 (1.0) zZ5.0, p!0.001

ACTH, adrenocorticotropic hormone; AUC, area under the curve.

Gender and HPA axis hormonal response
Figure 4 (a) Plasma ACTH response to naloxone by
gender in subjects who completed both the TSST and
naloxone challenge. Values reflect unadjusted means
(SE). Pl denotes time of placebo (saline) administration.
N denotes times of incremental naloxone administration.
*Plasma ACTH levels differed significantly by gender at
the following time points: 45 min, pZ0.013; 75 min, p!
0.001; 90 min, pZ0.008; 150 min, pZ0.021; 180 min, pZ
0.040. (b) Plasma cortisol response to naloxone by gender
in subjects who completed both the TSST and naloxone
challenge. Values reflect unadjusted means (SE). Pl
denotes time of placebo (saline) administration. N
denotes times of incremental naloxone administration.
*Plasma cortisol levels differed significantly by gender at
the following time points: 45 min, pZ0.015; 60 min, pZ
0.007; 75 min, pZ0.003; 90 min, p!0.001; 105 min, p!
0.001; 120 min, p!0.001; 135 min, pZ0.001; 150 min,
pZ0.001; 165 min, pZ0.001; 180 min, p!0.001.
higher cortisol response in female subjects to
naloxone compared to males. These results are in
agreement with HPA axis hormonal response to
other pharmacological challenges, including admin-
istration of CRH alone or combined with dexa-
methasone or arginine-vasopressin, which have
shown elevated HPA axis responsivity and
decreased feedback sensitivity in female subjects
compared to males (Gallucci et al., 1993; Heuser
649
et al., 1994; Born et al., 1995). However, following
a CRH challenge, other studies have reported no
significant gender difference in hormonal response
(Hermus et al., 1984).
The possible role of sex steroids such as estrogen
on the gender differences in response to the TSST
observed in our study is suggested by several lines of
evidence. In animal studies, estrogens enhance HPA
axis activity (Viau and Meaney, 1991; Burgess and
Handa, 1992) and, in response to stress females
have consistently shown greater increases in ACTH
and corticosterone compared with males (Kitay,
1961; Handa et al., 1994; Armario et al., 1995). In
humans, sex steroids seem to modulate the HPA
axis stress response as suggested by the observation
that cortisol responses to stress were similar in
males compared to females in the luteal phase of
the menstrual cycle whereas in the follicular phase
females had blunted response compared to males
(Kirschbaum et al., 1999). However, the direct
impact of estrogens on HPA axis regulation in
humans remains contradictory. Whereas in one
study a short-term estradiol application enhanced
cortisol responsivity to stress in males (Kirschbaum
et al., 1996), other studies show that the effect of
estrogen on stress reactivity is, if anything
reversed. For example, a 2-week estradiol treat-
ment in postmenopausal women did not modify
stress-induced HPA axis responses and feedback
sensitivity seemed to be increased resulting in a
blunted cortisol response to dexamethasone-CRH
test (Kudielka et al., 1999).
It is plausible that sex steroids may also influence
opioid regulation of HPA axis activation and con-
tribute to explain the gender differences in response
to naloxone observed in our study. The hypothalamic
CRH neurons receive direct inhibitory input from
b-endorphin-producing neurons located in the arc-
uate nucleus via the m-opioid receptor (Tsagarakis
et al., 1990). In addition, b-endorphin-producing
neurons inhibit norepinephrine neurons, which
provide direct stimulatory input to hypothalamic
CRH neurons (Jackson et al., 1990). There is prior
evidence that expression of m-opioid receptors is
modulated by gonadal steroid hormones in rat brain
(Hammer et al., 1994), as shown by increased
receptor binding to naloxone in hypothalamic
regions following exposure to estradiol (Brown
et al., 1996). Furthermore, the regional brain
content of opioid peptides is modulated by gonadal
steroid hormones; for example, in rodents estrogen
induces the expression and seems to stimulate the
release of endogenous opioid peptides that activate
opioid receptors in the limbic system and hypothala-
mus (Hammer et al., 1994; Priest et al., 1995;
Eckersell et al., 1998). It is plausible that if opioid
650
activity differs among genders, then HPA axis
activity would also differ as a function of gender
following naloxone administration.
The observation that acute psychological stres-
sors on one hand and pharmacological stimulation
tests on the other hand seem to result in different
gender-specific patterns of HPA axis responsivity is
intriguing. Reported gender differences could
possibly be attributed to differences in the applied
HPA axis stimulation procedures. In this regard
Stroud et al. (2002) observed that men and women
show different adrenocortical response to different
stressors; in their study, men showed greater
cortisol responses to achievement challenges, but
women showed greater cortisol responses to a
social rejection challenge. Thus, it appears that
different mechanisms of HPA axis activation stimu-
late the HPA axis in a singular way and are under
different neurochemical regulation. However, in
our study, the HPA axis stimulation procedures also
differed in their duration. For example, the
psychological stressor was administered over a 10-
min period and, differently, the naloxone challenge
was administered over a 2-h period. Thus, it is
possible that the gender differences observed in
response to naloxone administration could be
related to other mechanisms such as variation in
feedback regulation. In this regard, there is
evidence in preclinical models that estradiol
modulates mineralocorticoid (MR) and glucocorti-
coid (GR) receptors and thus modifies cortisol
negative feedback within the HPA axis (Peiffer
et al., 1991; Burgess and Handa, 1992; Handa et al.,
1994). Specifically, in estrogen treated rats, an
increase in the magnitude and the duration of the
corticosterone response to stress has been shown,
suggesting an impairment of the GR-mediated
negative feedback (Burgess and Handa, 1992).
Thus, the possibility that decreased cortisol
negative feedback in females as compared to
males could contribute to the increased hormonal
response observed following naloxone adminis-
tration cannot be ruled out. This observation may
reflect that, in addition to the nature of HPA axis
activation, the duration of the activation should
also be considered in future studies.
There is interest in investigating gender differ-
ences in HPA axis activation in response to
challenges as it may contribute to explain gender
differences in the prevalence of diseases associated
with HPA axis dysregulation. For example, gender
differences in HPA axis responses to stress may be
one mechanism underlying gender differences in
prevalence of depression (Boyd and Weissman,
1981; Stroud et al., 2002). Similarly, gender
differences in endogenous opioid activity could
M. Uhart et al.
play a role in the observed gender differences in
the development and maintenance of substance use
disorders such as alcohol dependence (Grant et al.,
2004; Oswald and Wand, 2004).
The present study has several weaknesses.
Although the sample size for the naloxone challenge
and TSST was ample, it would have been ideal if the
sample size was larger for the subgroup of subjects
that completed both the naloxone and TSST. The
moderate to large effect size suggests that this
would have resulted in finding significant gender
differences to the TSST. Second, it also needs to be
acknowledged that there are potential confounding
differences for participants in the between-subject
versus the within-subject portions of the study. For
the within-subject design, participants underwent
two challenges which activated the HPA axis. It is
plausible that a certain degree of desensitization
occurred between the first and second challenge as
subjects become accustomed to the stress of a
novel environment and intravenous line placement.
However, the wash out period between studies
should have minimized any significant desensitiza-
tion. Third, although race could have been a
confounder as previously described (Yanovski
et al., 2000), adjusting for race and other
demographic characteristics in our statistical
models likely mitigated bias induced by baseline
group differences. In addition, one should be
cautious about generalizing the pattern of
responses seen in artificial settings to real-life
situations. Furthermore, whether gender would
predict hormonal responses to other types of HPA
axis activators needs to be investigated. Further
research is needed to replicate the findings
reported here and to extend our understanding of
the underlying mechanisms for any gender differ-
ences in HPA axis hormonal response to challenge.
In summary, male subjects demonstrate a more
robust HPA axis hormonal response to a psychologi-
cal stressor compared to females and, in contrast,
females had greater hormonal reactivity to phar-
macological stimulation with naloxone compared to
males. Such gender differences are of interest as
potential contributors to gender differences in
health risks.
Acknowledgements
This work was supported by NIH grants AA 10158
(GSW), AA 12303 (GSW) and AA 12837 (MEM), and a
gift from the Kenneth A. Lattman Foundation
(GSW).
Gender and HPA axis hormonal response
References
Armario, A., Gavalda, A., Marti, J., 1995. Comparison of the
behavioural and endocrine response to forced swimming
stress in five inbred strains of rats. Psychoneuroendocrinology
20, 879–890.
Berger, M., Bossert, S., Krieg, J.C., Dirlich, G., Ettmeier, W.,
Schreiber, W., von Zerssen, D., 1987. Interindividual differ-
ences in the susceptibility of the cortisol system: an
important factor for the degree of hypercortisolism in stress
situations? Biol Psychiatry 22, 1327–1339.
Blevins Jr., L.S., Dobs, A.S., Wand, G.S., 1994. Naloxone-induced
activation of the hypothalamic–pituitary–adrenal axis in
suspected central adrenal insufficiency. Am. J. Med. Sci.
308, 167–170.
Born, J., Ditschuneit, I., Schreiber, M., Dodt, C., Fehm, H.L.,
1995. Effects of age and gender on pituitary–adrenocortical
responsiveness in humans. Eur. J. Endocrinol. 132, 705–711.
Boyd, J.H., Weissman, M.M., 1981. Epidemiology of affective
disorders. A reexamination and future directions. Arch. Gen.
Psychiatry 38, 1039–1046.
Brown, L.L., Pasi, S., Etgen, A.M., 1996. Estrogen regulation of
mu opioid receptor density in hypothalamic premammillary
nuclei. Brain Res. 742, 347–351.
Bucholz, K.K., Cadoret, R., Cloninger, C.R., Dinwiddie, S.H.,
Hesselbrock, V.M., Nurnberger Jr., J.I., Reich, T., Schmidt, I.,
Schuckit, M.A., 1994. A new, semi-structured psychiatric
interview for use in genetic linkage studies: a report on the
reliability of the SSAGA. J. Stud. Alcohol 55, 149–158.
Burgess, L.H., Handa, R.J., 1992. Chronic estrogen-induced
alterations in adrenocorticotropin and corticosterone
secretion, and glucocorticoid receptor-mediated functions
in female rats. Endocrinology 131, 1261–1269.
Calogero, A.E., 1995. Neurotransmitter regulation of the
hypothalamic corticotropin-releasing hormone neuron. Ann.
NY Acad. Sci. 771, 31–40.
Chrousos, G.P., 1997. Stressors, stress, and neuroendocrine
integration of the adaptive response. The 1997 hans selye
memorial lecture. Ann. NY Acad. Sci. 851, 311–335.
Cohen, J. 1988. Statistical power analysis for the behavioral
sciences, 2nd ed. Hillsdale, NJ.
Cohen, M.R., Pickar, D., Dubois, M., 1983. The role of the
endogenous opioid system in the human stress response.
Psychiatr. Clin. North Am. 6, 457–471.
Collins, A., Frankenhaeuser, M., 1978. Stress responses in male
and female engineering students. J. Human Stress 4, 43–48.
Eckersell, C.B., Popper, P., Micevych, P.E., 1998. Estrogen-
induced alteration of m-opioid receptor immunoreactivity in
the medial preoptic nucleus and medial amygdala.
J. Neurosci. 18, 3967–3976.
Frankenhaeuser, M., von Wright, M.R., Collins, A., von
Wright, J., Sedvall, G., Swahn, C.G., 1978. Sex differences
in psychoneuroendocrine reactions to examination stress.
Psychosom. Med. 40, 334–343.
Gallucci, W.T., Baum, A., Laue, L., Rabin, D.S., Chrousos, G.P.,
Gold, P.W., Kling, M.A., 1993. Sex differences in sensitivity of
the hypothalamic–pituitary–adrenal axis. Health Psychol. 12,
420–425.
Gianoulakis, C., 1998. Alcohol-seeking behavior: the roles of
the hypothalamic–pituitary–adrenal axis and the
endogenous opioid system. Alcohol Health Res. World 22,
202–210.
Gold, P.W., Chrousos, G.P., 2002. Organization of the stress
system and its dysregulation in melancholic and atypical
depression: high vs low CRH/NE states. Mol. Psychiatry 7,
254–275.
651
Grant, B.F., Dawson, D.A., Stinson, F.S., Chou, S.P.,
Dufour, M.C., Pickering, R.P., 2002. The 12-month preva-
lence and trends in DSM-IV alcohol abuse and dependence:
United States, 1991–1992 and 2001–2002. Drug. Alcohol
Depend. 74, 223–234.
Hammer Jr., R.P., Zhou, L., Cheung, S., 1994. Gonadal steroid
hormones and hypothalamic opioid circuitry. Horm. Behav.
28, 431–437.
Handa, R.J., Burgess, L.H., Kerr, J.E., O’Keefe, J.A., 1994.
Gonadal steroid hormone receptors and sex differences in
the hypothalamo–pituitary–adrenal axis. Horm. Behav. 28,
464–476.
Herman, J.P., Cullinan, W.E., 1997. Neurocircuitry of stress:
central control of the hypothalamo–pituitary–adrenocortical
axis. Trends Neurosci. 20, 78–84.
Hermus, A.R., Pieters, G.F., Smals, A.G., Benraad, T.J.,
Kloppenborg, P.W., 1984. Plasma adrenocorticotropin, corti-
sol, and aldosterone responses to corticotropin-releasing
factor: modulatory effect of basal cortisol levels. J. Clin.
Endocrinol. Metab. 58, 187–191.
Heuser, I.J., Gotthardt, U., Schweiger, U., Schmider, J.,
Lammers, C.H., Dettling, M., Holsboer, F., 1994. Age-
associated changes of pituitary–adrenocortical hormone
regulation in humans: importance of gender. Neurobiol.
Aging 15, 227–231.
Jackson, R.V., Grice, J.E., Jackson, A.J., Hockings, G.I., 1990.
Naloxone-induced ACTH release in man is inhibited by
clonidine. Clin. Exp. Pharmacol. Physiol. 17, 179–184.
Jessop, D.S., 1999. Review: central non-glucocorticoid inhibitors
of the hypothalamo–pituitary–adrenal axis. J. Endocrinol.
160, 169–180.
Kirschbaum, C., Wust, S., Hellhammer, D., 1992. Consistent sex
differences in cortisol responses to psychological stress.
Psychosom. Med. 54, 648–657.
Kirschbaum, C., Pirke, K.M., Hellhammer, D.H., 1993. The ‘Trier
Social Stress Test’—a tool for investigating psychobiological
stress responses in a laboratory setting. Neuropsychobiology
28, 76–81.
Kirschbaum, C., Klauer, T., Filipp, S.H., Hellhammer, D.H.,
1995a. Sex-specific effects of social support on cortisol and
subjective responses to acute psychological stress. Psycho-
som. Med. 57, 23–31.
Kirschbaum, C., Pirke, K.M., Hellhammer, D.H., 1995b. Prelimi-
nary evidence for reduced cortisol responsivity to psycho-
logical stress in women using oral contraceptive medication.
Psychoneuroendocrinology 20, 509–514.
Kirschbaum, C., Schommer, N., Federenko, I., Gaab, J.,
Neumann, O., Oellers, M., Rohleder, N., Untiedt, A.,
Hanker, J., Pirke, K.M., Hellhammer, D.H., 1996. Short-
term estradiol treatment enhances pituitary–adrenal axis and
sympathetic responses to psychosocial stress in healthy young
men. J. Clin. Endocrinol. Metab. 81, 3639–3643.
Kirschbaum, C., Kudielka, B.M., Gaab, J., Schommer, N.C.,
Hellhammer, D.H., 1999. Impact of gender, menstrual cycle
phase, and oral contraceptives on the activity of the
hypothalamus–pituitary–adrenal axis. Psychosom. Med. 61,
154–162.
Kitay, J.I., 1961. Sex differences in adrenal cortical secretion in
the rat. Endocrinology 68, 818–824.
Kreek, M.J., 1996. Opioid receptors: some perspectives from
early studies of their role in normal physiology, stress
responsivity, and in specific addictive diseases. Neurochem.
Res. 21, 1469–1488.
Kudielka, B.M., Hellhammer, J., Hellhammer, D.H., Wolf, O.T.,
Pirke, K.M., Varadi, E., Pilz, J., Kirschbaum, C., 1998. Sex
differences in endocrine and psychological responses to
652
psychosocial stress in healthy elderly subjects and the impact
of a 2-week dehydroepiandrosterone treatment. J. Clin.
Endocrinol. Metab. 83, 1756–1761.
Kudielka, B.M., Schmidt-Reinwald, A.K., Hellhammer, D.H.,
Kirschbaum, C., 1999. Psychological and endocrine responses
to psychosocial stress and dexamethasone/corticotropin-
releasing hormone in healthy postmenopausal women and
young controls: the impact of age and a two-week estradiol
treatment. Neuroendocrinology 70, 422–430.
Mangold, D., McCaul, M.E., Ali, M., Wand, G.S., 2000. Plasma
adrenocorticotropin responses to opioid blockade with
naloxone: generating a dose–response curve in a single
session. Biol. Psychiatry 48, 310–314.
McEwen, B.S., 1998. Protective and damaging effects of stress
mediators. N. Engl. J. Med. 338, 171–179.
Morley, J.E., Baranetsky, N.G., Wingert, T.D., Carlson, H.E.,
Hershman, J.M., Melmed, S., Levin, S.R., Jamison, K.R.,
Weitzman, R., Chang, R.J., Varner, A.A., 1980. Endocrine
effects of naloxone-induced opiate receptor blockade.
J. Clin. Endocrinol. Metab. 50, 251–257.
Oswald, L.M., Wand, G.S., 2004. Opioids and alcoholism. Physiol.
Behav. 81, 339–358.
Peiffer, A., Lapointe, B., Barden, N., 1991. Hormonal regulation
of type II glucocorticoid receptor messenger ribonucleic acid
in rat brain. Endocrinology 129, 2166–2174.
Petrie, E.C., Wilkinson, C.W., Murray, S., Jensen, C.,
Peskind, E.R., Raskind, M.A., 1999. Effects of Alzheimer’s
disease and gender on the hypothalamic–pituitary–adrenal
axis response to lumbar puncture stress. Psychoneuroendo-
crinology 24, 385–395.
Priest, C.A., Eckersell, C.B., Micevych, P.E., 1995. Estrogen
regulates preproenkephalin-A mRNA levels in the rat ventro-
medial nucleus: temporal and cellular aspects. Brain Res.
Mol. Brain Res. 28, 251–262.
Sapolsky, R.M., 2000. Glucocorticoids and hippocampal atrophy
in neuropsychiatric disorders. Arch. Gen. Psychiatry 57,
925–935.
Seeman, T.E., Singer, B., Charpentier, P., 1995. Gender
differences in patterns of HPA axis response to challenge:
macarthur studies of successful aging. Psychoneuroendocri-
nology 20, 711–725.
Seeman, T.E., Singer, B., Wilkinson, C.W., McEwen, B., 2001.
Gender differences in age-related changes in HPA axis
reactivity. Psychoneuroendocrinology 26, 225–240.
Sherwood Brown, E., Varghese, F.P., McEwen, B.S., 2004.
Association of depression with medical illness: does cortisol
play a role? Biol. Psychiatry 55, 1–9.
M. Uhart et al.
Stroud, L.R., Salovey, P., Epel, E.S., 2002. Sex differences in
stress responses: social rejection versus achievement stress.
Biol. Psychiatry 52, 318–327.
Traustadottir, T., Bosch, P.R., Matt, K.S., 2003. Gender
differences in cardiovascular and hypothalamic–pituitary–
adrenal axis responses to psychological stress in healthy
older adult men and women. Stress 6, 133–140.
Tsagarakis, S., Rees, L.H., Besser, M., Grossman, A., 1990.
Opiate receptor subtype regulation of CRF-41 release
from rat hypothalamus in vitro. Neuroendocrinology 51,
599–605.
Tsigos, C., Chrousos, G.P., 2002. Hypothalamic–pituitary–adrenal
axis, neuroendocrine factors and stress. J. Psychosom. Res.
53, 865–871.
Uhart, M., McCaul, M.E., Oswald, L.M., Choi, L., Wand, G.S.,
2004. GABRA6 gene polymorphism and an attenuated stress
response. Mol. Psychiatry 9, 998–1006.
Viau, V., Meaney, M.J., 1991. Variations in the hypothalamic–
pituitary–adrenal response to stress during the estrous cycle
in the rat. Endocrinology 129, 2503–2511.
Volavka, J., Cho, D., Mallya, A., Bauman, J., 1979. Naloxone
increases ACTH and cortisol levels in man. N. Engl. J. Med.
300, 1056–1057.
Wand, G.S., Mangold, D., El Deiry, S., McCaul, M.E., Hoover, D.,
1998. Family history of alcoholism and hypothalamic opioi-
dergic activity. Arch. Gen. Psychiatry 55, 1114–1119.
Wand, G.S., Mangold, D., Ali, M., 1999. Adrenocorticotropin
responses to naloxone in sons of alcohol-dependent men.
J. Clin. Endocrinol. Metab. 84, 64–68.
Wand, G.S., McCaul, M.E., Gotjen, D., Reynolds, J., Lee, S.,
2001. Confirmation that offspring from families with
alcohol-dependent individuals have greater hypothalamic–
pituitary–adrenal axis activation induced by naloxone
compared with offspring without a family history of alcohol
dependence. Alcohol Clin. Exp. Res. 25, 1134–1139.
Yanovski, J.A., Yanovski, S.Z., Boyle, A.J., Gold, P.W.,
Sovik, K.N., Sebring, N.G., Drinkard, B., 2000. Hypothala-
mic–pituitary–adrenal axis activity during exercise in African
American and Caucasian women. J. Clin. Endocrinol. Metab.
85, 2660–2663.
Yen, S., Jaffe, R., Barbieri, R., 1999. Reproductive endocrin-
ology: physiology, pathophysiology, and clinical manage-
ment. Saunders, Philadelphia, PA.
Zeger, S.L., Liang, K.Y., 1986. Longitudinal data analysis
for discrete and continuous outcomes. Biometrics 42, 121–
130.