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YHBEH-02985; No. of pages: 8; 4C:
Hormones and Behavior xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Hormones and Behavior
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h
Salivary testosterone does not predict mental rotation performance in men
or women
David A. Puts a,⁎, Rodrigo A. Cárdenas b, Drew H. Bailey c, Robert P. Burriss a, Cynthia L. Jordan b,d,
S. Marc Breedlove b,d
a
Department of Anthropology, Pennsylvania State University, University Park, PA 16802, USA
Department of Psychology, Michigan State University, East Lansing, MI 48824, USA
Department of Psychological Sciences, University of Missouri, Columbia, MO 65211, USA
d
Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA
b
c
a r t i c l e
i n f o
Article history:
Received 29 August 2009
Revised 3 March 2010
Accepted 4 March 2010
Available online xxxx
Keywords:
Androgens
Mental rotation
Sex difference
Spatial ability
Spatial cognition
Testosterone
a b s t r a c t
Multiple studies report relationships between circulating androgens and performance on sexually
differentiated spatial cognitive tasks in human adults, yet other studies find no such relationships. Relatively
small sample sizes are a likely source of some of these discrepancies. The present study thus tests for
activational effects of testosterone (T) using a within-participants design by examining relationships
between diurnal fluctuations in salivary T and performance on a male-biased spatial cognitive task (Mental
Rotation Task) in the largest sample yet collected: 160 women and 177 men. T concentrations were
unrelated to within-sex variation in mental rotation performance in both sexes. Further, between-session
learning-related changes in performance were unrelated to T levels, and circadian changes in T were
unrelated to changes in spatial performance in either sex. These results suggest that circulating T does not
contribute substantially to sex differences in spatial ability in young men and women. By elimination, the
contribution of androgens to sex differences in human performance on these tasks may be limited to earlier,
organizational periods.
© 2010 Elsevier Inc. All rights reserved.
Introduction
In animal models, adult behavioral sex differences have generally
been found to result from either the sex difference in circulating
androgens such as testosterone (T) in adulthood, or the sex difference
in exposure to androgens earlier in life, for example during the
perinatal period (Morris et al., 2004). These two modes of producing
behavioral sex differences have been described as “activational” and
“organizational,” respectively (Phoenix et al., 1959). Although there
are interesting exceptions to these two modes of differentiation, and
they often act through similar neural mechanisms (Arnold and
Breedlove, 1985), the dichotomy nevertheless holds true in many
cases.
Men and women perform similarly on tests of overall intelligence
but differ on tests that measure specific cognitive abilities (Hines,
2004). The largest cognitive sex differences are found in the domain of
spatial ability, with men tending to outperform women (Maccoby and
Jacklin, 1974). These differences may be due partly to lasting
organizational effects of prenatal or early postnatal androgens
⁎ Corresponding author. Fax: +1 814 863 1474.
E-mail address: dap27@psu.edu (D.A. Puts).
(Collaer et al., 2009; Puts et al., 2008), and/or pubertal androgens
(Hier and Crowley, 1982). Some human and rodent evidence suggests
a curvilinear relationship between androgen signaling and spatial
ability, such that organizational effects of androgens improve
performance on some spatial tasks in females and impair performance
on these tasks in gonadally intact males (Puts et al., 2007).
Androgens may also have transient activational effects on spatial
ability in adults, but evidence for this is equivocal. Several studies
have found significant relationships between T levels and spatial
ability in between-participants comparisons of adults (e.g., Christiansen, 1993; Christiansen and Knussmann, 1987; Driscoll et al., 2005;
Gordon and Lee, 1986; Hausmann et al., 2009, 2000; Hooven et al.,
2004; Moffat and Hampson, 1996a; Silverman et al., 1999), although
others have not (e.g., Falter et al., 2006; Halari et al., 2005; Hassler et
al., 1992; Janowski et al., 1998; Kampen and Sherwin, 1996; Matousek
and Sherwin, 2010; McKeever and Deyo, 1990) (see Table 1 and
Discussion). Moreover, correlations between adult androgen levels
and spatial performance in between-participants studies leave
questions about when during development androgen affects spatial
ability. Testosterone production rate is highly heritable (Meikle et al.,
1988), and it is therefore possible that intrasexual differences in
circulating T persist throughout life. If so, associations between adult
androgen levels and spatial ability may reflect prior organizational
effects of hormones.
0018-506X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.yhbeh.2010.03.005
Please cite this article as: Puts, D.A., et al., Salivary testosterone does not predict mental rotation performance in men or women, Horm.
Behav. (2010), doi:10.1016/j.yhbeh.2010.03.005
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Table 1
Results of previous studies that have investigated between-subjects relationships between spatial ability and T. ‘0’ indicates no relationship, ‘+’ and ‘−’ indicate positive and
negative relationships, respectively. ‘NL’ indicates a nonlinear relationship (quadratic or third-order polynomial).
Study
Sample size
Mean age
(SD, range)
Sample Samples,
method sessions
Alexander et al. (1998) 33M, 10M
41.1 (20–59); 33.4 (21–44) Blood
Burkitt et al. (2007)
39F, 36M
19.9 (2.8)
Saliva
Christiansen and
Knussmann (1987)
110M
24.1 (20–30)
Blood
Saliva
Christiansen (1993)
114M
26.4 (4.7, 18–38)
Driscoll et al. (2005)
Falter et al. (2006)
16F, 16M
22F, 24M
20–over 60
(19–41)
34F, 35M
F:24.1(4.6); M:24.1 (3.0)
Gordon and Lee (1986) 32M
Gouchie and Kimura
(1991)
Halari et al. (2005)
46F, 42M
Hassler et al. (1992)
25F, 26M
(18–35)
Blood
Saliva
Saliva
Saliva
Blood
“Multiple,” MRT
1
Surface development
Paper folding
Hidden patterns
2, 1
MRT
Virtual Morris water task
1, 2
Block design
Leistungsprufsystem subtest
Leistungsprufsystem subtest
1, 2
Block design
Leistungsprufsystem subtest
Leistungsprufsystem subtest
1, 1
Dichaptic stimulation test
1, 1
Dichaptic stimulation test
1, 1
Virtual Morris water task
1, 1
MRT
Targeting
Perceptual discrimination
Disembedding
11, 2
F:21.5 (18–31); M:21.0
(18–27)
F:27.69, (3.96); M:28.31
(4.81)
Saliva
2, 1
Blood
1, 1
F:18.77 (1.42); M:19.16
(1.65)
Blood
1, 1
Hausmann et al. (2000) 12F
29.1 (4.4, 23–38)
Blood
2, 1
Hausmann et al. (2009) 51F, 45M
Hooven et al. (2004)
28M
Janowski et al. (1998) 17F, 29M
23.4 (4.7), 25.8 (7.2)
23 (4)
F:29.8 (3.2, 24–34); M:28.5
(3.1, 23–34)
21.1 (18–29)
Saliva
Saliva
Blood
1, 1
1, 2
2, 2
Blood
1, 1
21.65 (0.92)
68.6 (4.4)
Urine
Blood
1, 2
1, 1
42F, 41M
58M
Unstated
Undergraduates
Blood
Blood
1, 1
4, 1
40F, 40M
F:23.0 (4.09); M:21.8
(2.35)
F:28.75 (19–43); M:28.6
(18–51)
24.5 (16–41)
24.5 (16–41)
Saliva
2, 1
Saliva
1, 1
Blood
Blood
1, 1
1, 1
22.42 (3.02)
(25–35), (60–80)
Saliva
Blood
2, 2
1, 2
42F, 41M
Kampen and Sherwin 32M
(1996)
Klaiber et al. (1967)
50M
Matousek and Sherwin 54M
(2010)
McKeever et al. (1987)
McKeever and Deyo
(1990)
Moffat and Hampson
(1996a)
Neave et al. (1999)
25F, 33M
Shute et al. (1983) 1
Shute et al. (1983) 2
48F, 43M
12F, 12M
Silverman et al. (1999) 59M
Young et al. (2010)
26 young M, 62
old M
Task
8
9
8
9
Cognitive laterality battery
– Localization
– Orientation
0
0
0
0
+
F:+, M:0
0
+
0
0
0
0
+
+
F:0, M:+
0
0
0
F:+ for 1 of 2 tasks,
M:0
– Touching blocks
– Form completion
Paper folding
MRT
MRT
Computerized judgment of line orientation
Modified judgment of line orientation
Spatial relations test
Hidden patterns
Dichaptic stimulation test
MRT
Mirrors pictures test
Hidden figures test
MRT
MRT
Block design task
Card rotation
MRT
0
Session 1:+,
Session 2:0
0
0
0
0
0
0
0
0
0
0
+
0
0
F:0, M:+
+
0
0
0
Block design task
MRT
Paper folding
Water level test
Block design test
Stafford identical blocks test
Stafford identical blocks test
Minnesota Paper Forms Board
MRT
Paper folding
MRT
−
0
0
0
0
0
0
0
F:0, M:NL
0
NL
French Reference Kit for Cognitive Factors — spatial tests
Minnesota Paper Forms Board
Primary mental abilities test/comprehensive ability
battery space test
MRT
MRT
F:0, M:NL
0
0
Figure discrimination task
Causal relationships are best tested by hormonal manipulation.
Demonstrating that androgen treatment elicits a particular behavioral
change, and that removal of treatment abolishes this effect,
constitutes strong evidence for activational effects of the hormone.
Several studies have reported activational effects of androgens on
Results
+
Young M:0, old M:
+
0
spatial task performance, but those that are placebo-controlled often
fail to demonstrate significant effects (reviewed in Puts et al., 2007).
Furthermore, these studies are frequently carried out using small and
possibly unrepresentative clinical samples, such as hypogonadal
males, Alzheimer patients, Turner Syndrome patients, and female-
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to-male transsexuals (Aleman et al., 2004; Alexander et al., 1998;
Cherrier et al., 2005; O'Connor et al., 2001; Ross et al., 2002; Van
Goozen et al., 1995; van Goozen et al., 2002; Wolf et al., 2000).
Within-participants correlational studies offer an alternative
means of assessing whether T has activational effects on spatial
performance. These studies can be carried out on larger, non-clinical
samples and have the power to show relationships between intraindividual changes in T and spatial performance. Within-participants
designs produce lower error variance due to variability among
individuals (i.e., differences among participants can be measured
and separated from error); consequently, within-participants designs
have greater statistical power and require fewer participants to show
significant relationships. Correlations may not demonstrate causation,
but if a causal relationship exists, then the putative dependent and
independent variables should be correlated. Thus, if T has purely
activational effects on spatial ability, then there should be correlations
between intra-individual fluctuations in T levels and spatial performance in adults.
Using a between-participants design, Moffat and Hampson
(1996a) found circadian changes in spatial performance that differed
significantly by sex. Males tended to improve over the morning,
whereas females exhibited the opposite trend. Because T levels also
tend to decrease over the morning in both sexes, Moffat and Hampson
suggested that the sex difference in performance change was the
result of high morning T levels augmenting female spatial ability and
impairing it in males. This hypothesis would be bolstered by evidence
for within-participants correlations between changes in T levels and
changes in spatial performance. However, the only previous study to
examine these correlations (Silverman et al., 1999) found no
significant relationship between changes in 59 men's T levels and
changes in their 3D mental rotation performance over a 12-hour
period.
In sum, the evidence is equivocal as to whether T has activational
effects on spatial ability in humans, and whether differences in adult T
levels might help explain some sex differences in spatial ability.
Relatively small sample sizes may account for some of the highly
varied results. We therefore sought to examine relationships in both
sexes between salivary T levels and performance on a spatial task that
shows one of the largest known human cognitive sex differences, the
3D Mental Rotation Task (Vandenberg and Kuse, 1978), in by far the
largest sample of men and women yet recruited. If testosterone has
activational effects on spatial ability, then:
1) Between-individual (“trait”) differences in circulating T levels will
predict differences in spatial performance.
2) Within-individual (“state”) changes in T levels will predict
changes in spatial performance.
Methods
Participants
Three hundred thirty-seven Michigan State University students
participated in this human subject board-approved study. Participants
were 160 women (20.43 yrs ± 1.53) and 177 men (20.14 yrs ± 1.71).
Participants' reported ethnicities were 91.4% White, 3.6% Asian, 2.1%
Hispanic or Latino, 1.2% Black or African American, 0.6% American
Indian or Alaska Native, and 1.2% reported identifying with another
ethnicity.
Scheduling of sessions
Participants were scheduled to participate in both a morning and
an evening session, approximately one week apart (6.99 ± 0.72 days).
Because the purpose of this study was in part to examine circadian
changes in T levels, it was desirable to minimize menstrual cycle-
3
related hormonal changes in women, which may affect spatial
performance (Hampson, 1990a; Hampson, 1990b; Hausmann et al.,
2000; Phillips and Silverman, 1997). Therefore, only women who
reported currently taking hormonal contraception participated in the
present study. We randomly allocated participants to attend their first
session during the morning or the evening, with their second session
taking place at the other time of day. Morning sessions began between
0820 h and 1000 h, and evening sessions began between 1720 h and
1900 h. An effort was made to maintain a consistent interval between
morning and evening sessions of nine hours, so participants who were
scheduled in the latter half of the morning testing session were also
scheduled in the latter half of the evening session, and vice versa. The
average time difference between the scheduled start times of the
morning and evening sessions was 8.95 h (±0.55). Each session
lasted approximately 1 h and included anthropometric and psychometric portions, separated by saliva collection. Anthropometric data
were collected for use in other studies that are not reported here.
Saliva collection
To minimize contamination of saliva samples, participants were
instructed not to eat, drink (except plain water), smoke, chew gum, or
brush their teeth for 1 h before their scheduled session. Women
wearing lipstick were asked to remove it with a tissue. Participants
rinsed their mouths with water immediately before chewing a piece
of sugar-free Trident gum (inert in salivary hormone assays) to
stimulate saliva flow. Each participant then collected approximately
10 ml of saliva in a sodium azide-coated polystyrene tube, after which
the tube was capped and stood upright at room temperature for 18–
24 h to allow mucins to settle. Each tube was then frozen at − 20 °C
until hormone analysis.
Testosterone assays
We obtained salivary unbound (“free”) testosterone concentrations, which correlate strongly with serum concentrations (e.g.,
Baxendale et al., 1980; Wang et al., 1981, r = 0.81 and 0.94,
respectively). Testosterone radioimmunoassays (RIAs) were performed by an experienced RIA technician in the Salivary Radioimmunoassay Laboratory at the University of Western Ontario. Two
hundred sixty-six female saliva samples (160 from Session 1, 106
from Session 2) and 333 male samples (177 from Session 1, 156 from
Session 2) were analyzed.
Following a double ether extraction, all samples were assayed in
duplicate using a Coat-A-Count kit for total testosterone (Diagnostic
Products, Los Angeles, CA), modified for use with saliva (for details,
see Moffat and Hampson, 1996b). RIAs were performed separately for
men and women in two batches for each sex. Sensitivity was 5–10 pg/
ml, and the average intra-assay coefficient of variation was 6.3%.
Duplicate assay concentrations were highly correlated (morning:
r(297) = 0.97, p b 0.0001; evening: r(301) = 0.97, p b 0.0001; controlling
for sex, r(294) = 0.93, p b 0.0001 and r(298) = 0.91, p b 0.0001, for
morning and evening, respectively). Consequently, each participant's
duplicates were averaged for each session. If a value was below
detectable levels for one duplicate, the duplicate assay with a detectable
level was used without averaging. This was the case for one session for
two female participants.
Psychometric testing
Fully-automated questionnaires and tasks were administered to
participants via computer. These included the Vandenberg and Kuse
(1978) 3D Mental Rotation Task (MRT) and several questionnaires
and tasks administered for other studies. In the MRT, participants are
shown 2-dimensional line drawings of 3-dimensional block figures.
For each item, a target block figure is shown on the left, followed by
Please cite this article as: Puts, D.A., et al., Salivary testosterone does not predict mental rotation performance in men or women, Horm.
Behav. (2010), doi:10.1016/j.yhbeh.2010.03.005
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four similar figures on the right. The task is to select the two figures on
the right that represent the target figure rotated in space. Digital
images were courtesy of Michael J. Tarr, Brown University. For
example stimuli, see Shepard and Metzler (1971). One point was
assigned for each item only if both correct answers were selected,
because this method of scoring has been shown to capture the largest
sex difference (Voyer et al., 1995). The MRT consisted of two 4-minute
sections of 10 items each.
Handedness was measured using a questionnaire developed by
Peters (1998), which employs 5-point scales to assess the degree to
which respondents prefer the left or right hand for 22 activities (e.g.,
draw, comb hair, wave goodbye). Scores for each respondent are
summed across all 22 activities. Sexual orientation was assessed using
the sexual attraction dimension of the Kinsey Scale (Kinsey et al.,
1948), which employs a 7-point scale ranging from “I am attracted to
men only, never to women” to “I am attracted to women only, never
to men” to identify the statement that best describes the respondent's
sexual feelings at present.
Female participants were also asked which brand of hormonal
contraception they were currently using. One hundred fifty-one
women answered this open-ended question, reporting the use of one
of 30 brands. Nine women did not answer. The brands identified by
female participants were then split into 10 categories according to the
active chemicals (e.g., ethinyl estradiol and desogestrel vs. ethinyl
estradiol and norethindrone), for the examination of any relationships
between types of hormonal contraception and testosterone concentrations or spatial performance.
Data treatment
Testosterone concentrations exhibited leftward-skewed distributions. We corrected these asymmetries by log-transforming
T concentrations before statistical analysis (Drennan, 1996).
Results
Validity and reliability measures
Testosterone
Mean salivary T concentrations for women were 20.35 ± 1.02 pg/
ml (morning) and 14.76 ± 0.70 pg/ml (evening). For men, T concentrations were 109.17 ± 3.52 pg/ml (morning) and 76.21 ± 2.32 pg/ml
(evening) (Table 2). These values are comparable to salivary T
concentrations obtained in previous studies (e.g., Dabbs, 1990; Moffat
and Hampson, 1996a, b). T was higher in men than in women (mixed
model ANOVA with session order (morning or evening first) and sex
as between-participants factors and time of day (morning or evening)
as a repeated measure: main effect of sex: F(1,257) = 1125.8,
p b 0.0001) and declined over the day in both sexes (main effect of
time of day: F(1,257) = 174.6.1, p b 0.0001). Controlling for session
order, T levels were also highly correlated within participants across
sessions (r(261) = 0.91, p b 0.0001; controlling for sex, r(258) = 0.59,
p b 0.0001). That is, participants who had high T in their morning
session also tended to have high T in their evening session a week
apart (Fig. 1), conforming to the notion of stable individual “trait”
differences in circulating T (Dabbs, 1990).
Table 2
Salivary testosterone levels were higher in the morning than in the evening and higher
in men than in women.
Women
Mean (SE, range)
T levels (pg/ml)
Men
AM
(N = 134)
PM
(N = 131)
AM
(N = 163)
PM
(N = 170)
20.35 (1.02,
4.5–67.5)
14.76 (0.70,
3.0–34.5)
109.17 (3.52,
28.0–301.5)
76.21 (2.32,
22.0–225.5)
Fig. 1. Within-subject relationships between (a) morning and evening salivary
testosterone levels and (b) Session 1 and Session 2 mental rotation test (MRT)
performance in women and men. Testosterone levels correlated strongly across
sessions, as did MRT scores.
Spatial ability
Session 1 mean MRT was 13.6 (±4.4) for men and 11.0 (±4.6) for
women out of a possible score of 20. These scores are intermediate
between the scores of 12.5 (men) and 10.3 (women) as reported by
Burkitt et al. (2007) and those of 15.9 (men) and 11.7 (women) as
reported by Burton et al. (2005), who gave 12 questions (rather than
10) per four-minute section. The decreased speed required to
complete the 10 items per section in the present study should have
reduced errors, raising scores, but the decreased number of possible
correct answers should have had the opposite effect on scores. Thus,
the MRT scores obtained in the present study are within the range
expected from this participant population, given the design and
scoring of the test.
Men's scores on the MRT were on average higher than women's
(mixed model ANOVA with session order (morning or evening first)
and sex as between-participants factors and testing session as a
repeated measure: main effect of sex: F(1,300) = 21.8, p b 0.0001).
MRT performance also improved from Session 1 to Session 2 (main
effect of testing session: F(1,300) = 103.1, p b 0.0001; interaction
with sex: F(1,300) = 1.53, p = 0.22). MRT scores did not differ
between morning and evening sessions (main effect of session time
(morning or evening): F(1,301) = 0.57, p = 0.45; interaction with
sex: F(1,301) = 1.74, p = 0.19). Controlling for session order, MRT
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Behav. (2010), doi:10.1016/j.yhbeh.2010.03.005
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5
scores were also highly correlated within participants across
sessions (r(301) = 0.75, p b 0.0001; controlling for sex, r(300) = 0.73,
p b 0.0001) (Fig. 1).
Hormonal contraception
The combination of active ingredients in the hormonal contraception used by the participants was unrelated to salivary T levels or
spatial ability. There were no statistically significant effects of
hormonal contraception active ingredients (10 categories) on
morning or evening T levels, MRT performance in either session
(one-way ANOVA with hormonal contraception type as predictor
variable (10 levels) and T levels or MRT scores as outcome variables),
or improvement across sessions in MRT (mixed model ANOVA with
MRT scores as within-participants repeated measures outcome
variable). Consequently, hormonal contraception type is not included
in the following analyses.
Relationships between T and mental rotation ability
Relationships between T and mental rotation ability were tested
using random-intercept multilevel models in SAS PROC MIXED (SAS
Institute, 2004).
Testosterone as a time-varying covariate
A model with testing session and T as predictors yielded
significant effects for testing session (t(165) = 8.06, p b 0.0001) and
T (t(165) = 3.32, p = 0.001). However, because T is so closely related
to sex (Fig. 1), and because sex is confounded by so many variables
other than current T levels, we measured within-sex effects of T by
inserting sex into this model. When sex and T are included in the
same model, the effect of T can be interpreted as within-sex variation
in T, and the effect of sex can be interpreted as a sex difference
adjusted for the sex difference in T. The model with testing session, T,
and sex as predictors yielded significant effects for testing session
(t(165) = 7.72, p b 0.0001) and sex (t(165) = 2.45, p = 0.02). However, the effect for T was no longer significant (t(165) = 0.41, p = 0.69).
When the T × sex interaction was added to this model, the interaction
was nonsignificant (t(164) = −0.13, p = 0.90), and the effect of T
remained nonsignificant (t(164) = 0.25, p = 0.80). Because these effects
are in opposite directions, the within-sex effect of T was nonsignificant
for both sexes (see also Fig. 2).
Within- and between-participants effects of testosterone
To test the hypothesis that between-participant differences in trait
levels of T and within-participant T fluctuations have different effects
on MRT performance, we partitioned T into a between-participants
component (i.e., mean T across testing sessions) and a withinparticipants component (i.e., the difference between Session 2 T and
Session 1 T). Both were treated as participant-level variables, meaning
that they did not differ across testing session.
If between-participant differences in T affect mental rotation, then
the between-participants variable should be significantly related to
performance on the MRT. If within-participant variations in T affect
spatial ability, then the within-participants variable should be
significantly related to performance on the MRT, but only in the
second testing session. Therefore, the within-participants component
of T was allowed to interact with the testing session variable. If the
within-participants variable is significantly related to performance on
the MRT, but only in the second testing session, then the withinparticipants parameter should not reach statistical significance, but
the interaction between the within-participants parameter and
testing session should.
The model with sex, test session, the between-participants
component of T, the within-participants component of T, and the
interaction between within-participants T and testing session
produced significant effects of sex (t(165) = 2.58, p = 0.01)
Fig. 2. Salivary testosterone levels and mental rotation test (MRT) performance scores
were related in neither (a) women nor (b) men.
and testing session (t(165) = 7.66, p b 0.0001). The effects of the
between-participants component of T (t(165) = −1.21, p = 0.23), the
within-participants component of T (t(165) = −0.81, p = 0.40),
and the interaction between within-participants T and testing session
(t(165) = 0.45, p = 0.66) were not statistically significant.
Testosterone and spatial learning
To test the hypothesis that higher levels of T during the first testing
session led to an increased proficiency on the MRT in the second
testing session, Session 1 T was transformed into an individual level
variable. If higher levels of T during the first testing session affect
subsequent, but not current, performance on the MRT, then Session 1
T should interact significantly and positively with testing session.
The model with Session 1 T, sex, testing session, and the
interaction between Session 1 T and testing session yielded a
significant effect for testing session (t(193) = 8.70, p b 0.0001)
but no significant effects for sex (t(193) = 1.74, p = 0.08), Session 1
T (t(193) = 1.20, p = 0.23), or the interaction between Session 1 T and
testing session (t(193) = −1.94, p = 0.054). While the interaction
falls near the cutoff for statistical significance, it is in the opposite
direction of what we would predict based on the hypothesis that
higher levels of T during the first testing session led to an increased
proficiency on the MRT in the second testing session.
To examine whether collinearity between testosterone and testing
session made it impossible to simultaneously observe the effects of
testosterone and session on MRT, we calculated a model with sex and
testosterone as the only predictors of MRT performance. The effect of
sex remained significant (t(166) = 3.28, p = 0.001), and the effect of
testosterone remained nonsignificant (t(166) = −0.53, p = 0.59).
Please cite this article as: Puts, D.A., et al., Salivary testosterone does not predict mental rotation performance in men or women, Horm.
Behav. (2010), doi:10.1016/j.yhbeh.2010.03.005
ARTICLE IN PRESS
6
D.A. Puts et al. / Hormones and Behavior xxx (2010) xxx–xxx
Other potential relationships between testosterone and mental rotation
ability
It is possible that T has a more complex relationship with mental
rotation ability than tested in the models above. Consequently, we
tested 24 additional models, including additional scorings and
scalings of the MRT, additional covariates, quadratic effects of T, and
higher-order interactions. All of the models above containing sex as a
predictor were rerun using an alternate scoring of the MRT (scoring
correct answers without requiring both correct answers for each item,
for a possible 40 points) as the dependent variable (see, e.g., Moffat
and Hampson, 1996a). Secondly, all of the models were rerun using
the squares of the first and second scorings of the MRT as dependent
variables. Lastly, all of the models using both scorings and scalings of
the MRT were rerun with T squared as an additional predictor, testing
interactions between this variable and all variables with which T was
tested to interact. In none of these models were T, its quadratic
component, or any interactions containing T found to significantly
predict MRT score, except for one parameter. After controlling for the
quadratic component of Session 1 T and its interaction with session, a
significant negative effect for the interaction between Session 1 T and
session was observed (t(191) = −2.08, p = 0.04). However, this
finding – which falls on the border of statistical significance – is
only observed using this particular scoring and scaling of the MRT and
after controlling for the quadratic component of Session 1 T and its
interaction with session, and is inconsistent with the prediction that
higher Session 1 T improves spatial learning.
Entering handedness and sexual orientation as control variables
into analyses did not alter any of the above results.
Discussion
The present study is the first to examine within-participant diurnal
changes in T and explore their possible relationship to spatial
performance in women, and only the second to do so in men. This
study also offers by far the largest sample to examine relationships
between T concentrations and spatial ability within- or betweenparticipants. T levels were unrelated to performance on the Vandenberg and Kuse 3D Mental Rotation Task, a male-biased spatial task
showing one of the largest known human cognitive sex differences.
Individual differences in T levels did not predict individual differences
in mental rotation performance in either sex. Furthermore, the
between-session improvement in performance was unrelated to T
levels, and circadian changes in T were unrelated to changes in
performance in either sex.
There are multiple reasons to believe that these findings are valid.
First, T levels and mental rotation performance showed the normative
sex differences (Dabbs, 1990; Vandenberg and Kuse, 1978; Voyer et
al., 1995), and T levels exhibited expected diurnal changes and were
highly correlated within participants across sessions (Dabbs, 1990).
Second, the present study employed a far larger sample than those of
previous studies, both in terms of numbers of participants and total T
samples analyzed. Third, although power calculations are inappropriate in the interpretation of results (Hoenig and Heisey, 2001), test
statistics for relationships between T levels and spatial performance
were generally near zero and did not approach threshold for rejecting
the null hypothesis. Finally, to maximize the chance of finding
significant relationships, we did not correct for multiple statistical
tests when exploring additional statistical models to our main
analysis. Had we done so, relationships between T levels and mental
rotation performance would have been even further from achieving
statistical significance.
We attempted to minimize the influence of menstrual cycle phase
on spatial performance and sex hormone levels by examining only
female participants who were taking hormonal contraception.
Although we view this as an advantage of our methods, this approach
may be viewed as a limitation because it supports conclusions only
about women taking hormonal contraception. Furthermore, although
we found no effects of the type of hormonal contraception that our
participants used on T levels or MRT performance, sex steroids may
have fluctuated in women depending on whether they were in the
“on” versus “off” phase of hormonal contraception use, introducing
noise. However, such noise seems unlikely to have contributed
significantly to our failure to find significant relationships between T
levels and MRT performance, given that relationships between
salivary T and mental rotation ability were found in neither sex.
Moreover, although hormonal contraception and other factors such as
diurnal fluctuations in cortisol and glucose levels could have
introduced noise, reducing our ability to detect relationships between
T and MRT performance, the influence of this noise should have been
reduced by our large sample size and our use of both between- and
within-participants designs.
We also analyzed possible confounding effects of handedness and
sexual orientation and found no significant relationships between T
and MRT performance or learning after controlling for these variables
and their interactions. However, it remains possible that T could affect
mental rotation in interaction with other hormones and nonhormonal factors that we did not examine.
It is also possible that a different male-biased spatial test from the
Vandenberg and Kuse mental rotation test, or an alternative method
of administering this test, would produce significant relationships
between performance and salivary T concentrations. For example,
perhaps the easier task implemented in the present study (10
questions and 4 min per section versus other methods, e.g., 12
questions and 3 min per section) might lead to scores that are less
susceptible to changes in T levels. While we cannot rule out this
possibility, the task used in the present study showed a large, highly
robust sex difference, and the mental rotations test is the most
frequently used test in studies of sex differences in spatial ability (e.g.,
Table 1 in Voyer et al., 1995). Scores on this test also correlate with
performance on other male-biased spatial tasks, for example real
world (Silverman et al., 2007) and virtual (Driscoll et al., 2005)
navigation. Thus, this test constitutes one of the best candidates for
showing a relationship between spatial performance and salivary T.
Although we employed only one method of task administration (10
questions and 4 min per section), we examined two methods of
scoring (see above) and found relationships between salivary T and
performance using neither scoring method.
We collected two saliva samples per participant and one sample
per session. Because T secretion is pulsatile, this method will have
limited our ability to detect individual differences and withinindividual diurnal variation in T production. However, morning and
evening T levels were highly correlated across participants, even
within sexes, suggesting that we captured a substantial proportion of
the between-individual variation in T production. Furthermore,
T levels showed a highly significant drop from morning to evening
sessions, indicating that we were able to capture a substantial
proportion of the diurnal variation in T production.
Our final caveats regard the representativeness of our sample. We
examined only a restricted age range of young adults, and so it is
possible that different results might have been obtained had we
examined other ages (see below). In addition, there was notable
attrition from the first to second sessions, especially in women (54
women versus 21 men, see Methods). This sex difference may be due
to the greater difficulty reported by women in collecting the nearly
10 ml of saliva required. However, we can think of no reason why the
relationship between T and spatial performance would differ between
those who continued and those who dropped out of our study.
Overall, our results suggest that any within-individual effects of
changing T levels would have to occur on a time scale longer than the
diurnal intervals examined in this study and that of Silverman et al.
(1999). Although Moffat and Hampson (1996a) found circadian
changes in mental rotation performance – as did Sanders et al. (2002),
Please cite this article as: Puts, D.A., et al., Salivary testosterone does not predict mental rotation performance in men or women, Horm.
Behav. (2010), doi:10.1016/j.yhbeh.2010.03.005
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D.A. Puts et al. / Hormones and Behavior xxx (2010) xxx–xxx
though only in men, and in only one of two samples – neither
Silverman et al. (1999) nor the present study replicated these results.
Furthermore, none of these studies found relationships between
diurnal changes in T and changes in spatial performance.
Spatial ability could change as a result of longer-term variations in
T levels, such as those due to menstrual cycle phase or season.
Hausmann et al. (2000) found a relationship between mental rotation
performance and the T levels of 12 women sampled at several times in
their cycle. Kimura and Hampson (1994) found seasonal changes in
spatial performance in young men, which the authors suggested may
have resulted from fluctuating T levels. These changes may be
analogous to those observed in meadow voles (Microtus pennsylvanicus) and deer mice (Peromyscus maniculatus), in which males
expand their home ranges during the breeding season in order to
increase access to mates (Galea et al., 1996, 1995; Gaulin and
FitzGerald, 1989). In both of these species, males outperform females
in laboratory spatial tasks only during the breeding season (Galea et
al., 1996; Gaulin and FitzGerald, 1989) when T levels are elevated
(Galea and McEwen, 1999).
However, other cyclically fluctuating factors could cause menstrual cycle and seasonal variation in human spatial ability, and the
aggregate of previous findings suggests a weak, if any, causal
relationship between levels of circulating T and spatial ability in
adults. For example, Halari et al. (2005) found no between-individual
relationships between T and mental rotation performance, and while
Moffat and Hampson (1996a) found a significant negative linear
relationship in men, Silverman et al. (1999) found a significant
positive relationship. Driscoll et al. (2005) also found a positive
correlation between salivary T and virtual water maze performance in
men but not in women. This relationship was found only with
both young and elderly men included in the analysis (not in either
group separately), so it is possible that declining T levels were
tracking the decline of some other function or functions associated
with spatial cognition. This interpretation is supported by the finding
of Young et al. (2010) that mental rotation performance correlated
with T levels in older men but not in younger men. Moreover, in the
present study, by far the largest of its kind, spatial performance was
not associated with T levels in between-participants comparisons. It is
possible that previously reported associations between spatial
performance and adult T concentrations reflect prior organizational
effects of T and a tendency for adult levels to correlate with earlier
ones.
Further, we suggest that testosterone treatment studies generally
find negligible effects on spatial ability. Although some studies have
claimed to show effects of T treatment on spatial performance (e.g.,
Van Goozen et al., 1994), those that used placebo-treated controls
often find no improvement beyond normal learning effects. For
example, Alexander et al. (1998) found no effect of six weeks of T
treatment on spatial performance in 33 hypogonadal and 10
eugonadal men, ages 21–59. Young et al. (2010) found no effect of
six weeks of T treatment on spatial performance in 32 older and 13
younger men. O'Connor et al. (2001) found no effect of eight weeks of
T treatment on spatial performance in seven hypogonadal men (ages
23–40), although performance in 15 eugonadal men failed to exhibit
normal practice effects after T treatment. Similarly, Aleman et al.
(2004) found improvement (likely due to practice) in 12 women
treated with placebo on their first testing session and T on their
second, but no improvement in 14 women treated with T then
placebo. Van Goozen et al. (2002) found no effects of T treatment in 19
female-to-male transsexuals on several male-biased spatial tasks,
including mental rotation. Likewise, Ross et al. (2003) observed no
improvement in spatial abilities in 26 androgen-treated Turner
Syndrome (TS) patients relative to placebo-treated TS controls. Wolf
et al. (2000) found no effect on spatial ability of a single T injection
relative to placebo in 30 elderly men. Although several studies have
found cognitive improvements following T treatment in elderly men
7
(Cherrier et al., 2001; Janowsky et al., 2000, 1994), including
Alzheimer patients (Cherrier et al., 2005), these effects were not
restricted to spatial tasks or to tasks showing a male advantage and
seemed only to restore practice effects present in younger men. Thus,
these treatment effects do not provide evidence that circulating
T contributes to spatial cognitive sex differences in younger adults.
Finally, putative activational effects of testosterone on spatial
ability may be questioned on theoretical grounds (Puts et al., 2007).
Spatial behaviors and their underlying neural systems should remain
susceptible to hormonal fluctuations only if the cost of maintaining
such plasticity was counterbalanced by fitness benefits over the
evolutionary history of the organism. This is likely to occur if spatial
demands change significantly and repeatedly (e.g., seasonally). In
some rodent species, males expand their home ranges during the
breeding season in order to increase access to mates, and males
outperform females in laboratory spatial tasks only during the
breeding season when T levels are elevated (see above). In relatively
non-seasonal species, such as laboratory rats, spatial ability appears to
be comparatively unresponsive to T after certain early critical periods
(Commins, 1932). The aggregate of human research seems to suggest
that, at least within the normal range of circulating levels in young
adults, T has little activational effect on performance on male-biased
spatial tasks. Given that humans exhibit very low breeding seasonality, these findings might be expected.
If, as our data indicate, circulating T has little effect on spatial
cognitive performance in young men and women, then the well
established sex differences in such performance cannot be attributed
to the sex difference in circulating T. By elimination, this would
suggest that any role for androgens in establishing sex differences in
spatial ability must occur earlier in life, either perinatally or
pubertally, when reliable sex differences in androgen secretion
could have lasting, organizational effects.
Acknowledgments
We thank Bradly Alicea, Michael Burla, Melina Durhal, Rebecca
Frysinger, Sana Khan, Mallory Leinenger, Erin MacCourtney, Heather
Malinowski, Ernestine Mitchell, Joe Morehouse, Sara Sutherland, Lisa
Vroman, Tyesha Washington, and Molly Zolianbawi for their
conscientious assistance in study preparation and data collection,
and Elizabeth Hampson and Bavani Rajakumar for their assistance
with hormone assays.
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