Arch Sex Behav (2009) 38:6–15
DOI 10.1007/s10508-007-9291-z
ORIGINAL PAPER
Prenatal Sex Hormones (Maternal and Amniotic Fluid)
and Gender-related Play Behavior in 13-month-old Infants
Cornelieke van de Beek Æ Stephanie H. M. van Goozen Æ
Jan K. Buitelaar Æ Peggy T. Cohen-Kettenis
Received: 28 June 2006 / Revised: 8 October 2007 / Accepted: 28 October 2007 / Published online: 13 December 2007
Ó Springer Science+Business Media, LLC 2007
Abstract Testosterone, estradiol, and progesterone levels
were measured in the second trimester of pregnancy in
maternal serum and amniotic fluid, and related to direct
observations of gender-related play behavior in 63 male and
63 female offspring at age 13 months. During a structured
play session, sex differences in toy preference were found:
boys played more with masculine toys than girls (d = .53)
and girls played more with feminine toys than boys (d =
.35). Normal within-sex variation in prenatal testosterone
and estradiol levels was not significantly related to preference for masculine or feminine toys. For progesterone, an
unexpected significant positive relationship was found in
boys between the level in amniotic fluid and masculine toy
preference. The mechanism explaining this relationship is
presently not clear, and the finding may be a spurious one.
The results of this study may indicate that a hormonal basis
for the development of sex-typed toy preferences may
manifest itself only after toddlerhood. It may also be that
C. van de Beek (&)
Department of Child and Adolescent Psychiatry, VU University
Medical Center, Postbus 303, 1115 ZG Duivendrecht,
Amsterdam, The Netherlands
e-mail: [email protected]
S. H. M. van Goozen
School of Psychology, Cardiff University, Cardiff, UK
J. K. Buitelaar
Department of Psychiatry, St. Radboud University Nijmegen
Medical Center, Nijmegen, The Netherlands
P. T. Cohen-Kettenis
Department of Medical Psychology, VU University Medical
Center, Amsterdam, The Netherlands
123
the effect size of this relationship is so small that it should
be investigated with more sensitive measures or in larger
populations.
Keywords Sex hormones Maternal hormones Amniotic fluid Play behavior Gender role Sex differences
Introduction
Toy preference is one of the earliest manifestations of gender-related behavior. Girls have been found to prefer playing
with toys such as dolls and household supplies, whereas
boys prefer playing with toys such as vehicles and weapons
(Hines, 2004). Several studies have demonstrated that sex
differences in play behavior are present in the second year of
life (e.g., Caldera, Huston, & O’Brien, 1989; Fagot, 1974;
O’Brien & Huston, 1985). A study by Servin, Bohlin, and
Berlin (1999) showed that girls and boys chose different toys
as early as the age of 12 months.
The fact that parents engage in some form of sex typing of
their infant’s play behavior in the first year of life (Fagot, 1995;
Fisher-Thompson, 1993; Lytton & Romney, 1991) suggests
that socializing influences already may play a role. However,
there are also indications that the child himself or herself may
have certain innate preferences, i.e., the tendency to be focused
on specific aspects of objects, like movement, color or form
(Alexander, 2003), which may prime them to prefer specific toy
categories. The findings of sex differences in toy preferences in
non-human primates (Alexander & Hines, 2002), in which the
influence of social learning and cognitive concepts or beliefs on
toy preference can be considered nil, give additional reason to
assume that there is a biological basis for the development of
sex-typed toy preferences.
Arch Sex Behav (2009) 38:6–15
Among the biological determinants of play behavior, sex
hormones are likely candidates. Prenatal sex hormones can
permanently influence postnatal human sex-typed behaviors
by altering fetal brain development (for review and discussion, see Berenbaum, 1998; Cohen-Bendahan, Van de Beek,
& Berenbaum, 2005; Collaer & Hines, 1995; Gooren &
Kruijver, 2002; Kawata, 1995). Moreover, since there is also
much within-sex variation in gender-typical behaviors, such
as tomboy behavior (Bailey, Bechtold, & Berenbaum, 2002;
Morgan, 1998), sex hormones are probably not only responsible for behavioral sex differences, but also for within-sex
variations in gender-related behavior.
Animal studies have shown that there are a few critical
periods in development in which the brain regions that are
responsible for the regulation of the sex-typed behavior are
highly sensitive to the effects of sex hormones (Hines, 2004).
In humans, the period between weeks 8 and 24 of gestation
may be particularly important for sexual differentiation
because this is the period when the male fetus shows a peak in
serum testosterone (at its highest at about week 16) (Smail,
Reyes, Winter, & Faiman, 1981).
It has been clearly shown that prenatal androgens can have
masculinizing and defeminizing effects on postnatal human
behaviors (Hines, 2004). Very little is known about the function
of ‘‘female-typical’’ hormones in sexual differentiation and
their effects on human behavioral differentiation. Although
rodent studies indicate that estradiol is very important in
behavioral masculinization (Collaer & Hines, 1995; Goy &
McEwen, 1980) and defeminization (Fitch & Denenberg,
1998), its role in humans is less obvious. Findings in both girls
and boys, exposed to diethystilbestrol (DES) (a synthetic
estrogen), suggest a general lack of influences on childhood
gender typical play (Hines, 2004).
Studies that have investigated the behavioral effects of
prenatal progestagens mostly focused on exogenously administered synthetic progestins. The interpretations of the findings
are complicated by the fact that different types of progestins
were used. Prenatal exposure of girls to androgen-based synthetic progestins seemed to result in more male-typical
behavior, such as more tomboyism and a stronger preference
for male-typical toys, whereas exposure of girls to progesterone-based synthetic progestins produced fewer and opposite
effects. Similar but less pronounced effects of these two types
of exogenous progestins were found in boys (for review, see
Collaer & Hines, 1995). Less is known about the effects of
normal circulating progesterone levels during pregnancy,
although an antiandrogenic effect on the fetus has been proposed (Ehrhardt, Meyer-Bahlburg, Feldman, & Ince, 1984).
Currently, most evidence of a prenatal effect of androgens
on human play behavior comes from studies in girls with
congenital adrenal hyperplasia (CAH). CAH is a genetic
condition that results in the production of high adrenal
androgens beginning very early in gestation. Girls with CAH
7
show both masculinization and defeminization of behavior:
they have a stronger preference for traditionally masculine
toys and activities than unaffected control girls (relatives or
matched comparisons) (Berenbaum & Hines, 1992; Pasterski et al., 2005). Indications for a dose–response relationship
have also been found: more severely affected girls with CAH
were more interested in masculine careers and toys than less
affected girls (Servin, Nordenström, Larsson, & Bohlin,
2003). Furthermore, the timing of exposure on behavior has
been demonstrated: prenatal androgen exposure, but not
early or later postnatal exposure, was related to more maletypical play behavior (Berenbaum, Duck, & Bryk, 2000). In
boys with CAH, elevated prenatal adrenal androgen exposure does not seem to result in more masculine play behavior
(Berenbaum & Hines, 1992).
There is also evidence that normal variations in androgen
levels are systematically related to early sex differences and
within-sex variations in gender-typical play behavior of
children without clinical conditions. This evidence comes
from studies using maternal blood to infer hormonal effects on
the fetus. Udry, Morris, and Kovenock (1995) found a relationship between prenatal maternal sex hormone binding
globulin (SHBG, which is responsible for binding testosterone
and therefore inversely related to the amount of biologically
active testosterone) and gender role behavior in 27- to 30year-old women. In correspondence with the idea that prenatal
sexual differentiation of the human brain takes place during
the testosterone peak, in the second rather than the first or third
trimester, maternal SHBG levels in the second trimester predicted gender role behavior. Unexpectedly, no significant
relationship was found between maternal testosterone and
the women’s postnatal gender role behavior. In a very large
sample, however, Hines, Golombok, Rust, Johnston, and
Golding (2002) found higher maternal testosterone levels, as
measured between weeks 5 and 36 of gestation, to be related to
more masculine-typical preschool activity interests (motherreported) in 3.5-year-old girls. This relationship was not found
in boys, which may indicate that the threshold for masculinization of behavior by androgens is exceeded in healthy males
such that variability within the normal range would not be
associated with individual differences in their gender role
behavior.
Both Hines et al. (2002) and Udry et al. (1995) used maternal blood as an indicator of prenatal androgen exposure of the
fetus. Recently, a positive correlation was found between
maternal and fetal blood testosterone (Gitau, Adams, Fisk, &
Glover, 2005). This suggests that testosterone may cross the
placenta, but it may also reflect a genetic relationship. However,
maternal androgens do not appear to come from the fetus, as
several studies have failed to find a difference in serum second
trimester testosterone levels between women carrying a male
and those carrying a female fetus (Forest, Ances, Tapper, &
Migeon, 1971; Glass & Klein, 1980; Meulenberg & Hofman,
123
8
1991; Rivarola, Forest, & Migeon, 1968; Van de Beek, Thijssen, Cohen-Kettenis, Van Goozen, & Buitelaar, 2004).
Another approach to investigate the organizational effects of
prenatal sex hormones is used by amniotic fluid studies.
Amniotic fluid can only be obtained from amniocentesis
conducted for purposes of diagnosing fetal anomalies. Coincidentally, this medical intervention usually takes place around
week 16 of gestation, a time that appears to correspond well to
the male testosterone peak. Amniotic fluid seems to provide
information about the sex steroid production by the fetus since
several studies have found large sex differences in amniotic
androgens (Dawood & Saxena, 1977; Finegan, Bartleman, &
Wong, 1989; Judd, Robinson, Young, & Jones, 1976; Nagamani, McDonough, Ellegood, & Mahesh, 1979; Robinson,
Judd, Young, Jones, & Yen, 1977; Rodeck, Gill, Rosenberg, &
Collins, 1985; Van de Beek et al., 2004). However, currently
there is no hard evidence of a direct relationship between
amniotic testosterone and fetal serum testosterone. The mostly
low and/or non-significant correlations between androgens
measured in amniotic fluid and in maternal serum suggest that
these measures reflect something different (Van de Beek et al.,
2004). In addition, there is also another methodological difference between these two measures: amniotic fluid measures
are only available for a specific population, which contains
generally older mothers, while maternal blood measures can be
relatively easily measured in a representative sample of the
general population. Therefore, both measures have their
advantages and disadvantages, and might provide convergent
evidence of the relationship between prenatal hormones and
postnatal behavior.
Until now, there are two groups of investigators that
have examined the relationship between early androgen exposure by investigating prenatal amniotic hormones and postnatal
behavior. One research group mainly looked at measures of
cognition and cerebral lateralization (Finegan, Niccols, &
Sitarenois, 1992; Grimshaw, Bryden, & Finegan, 1995;
Grimshaw, Sitarenios, & Finegan, 1995). The other group of
investigators primarily focused on aspects of early social
development (Knickmeyer, Baron-Cohen, Raggatt, & Taylor,
2005; Lutchmaya, Baron-Cohen, & Raggatt, 2001, 2002), but
also included one aspect of gender role behavior in their design,
namely game participation (Knickmeyer et al., 2005). They did
not find amniotic testosterone to be related to individual differences on the Masculinity and Femininity scales of the
Children’s Play Questionnaire at age 5/6 years, despite the fact
that this measure showed large sex differences in the same
sample. However, this parent report instrument might not have
been sufficiently sensitive to within sex variability in behavior,
as opposed to between-sex differences. Also, the very small
sample size may have increased the chance of negative results.
In the current study, we focused on toy preference observed
in a laboratory situation. Rather than using parental reports,
play behavior was scored by trained observers. Our first goal
123
Arch Sex Behav (2009) 38:6–15
was to replicate previously reported sex differences in masculine and feminine toy preference in normally developing
13-month-old infants. Furthermore, we investigated whether
prenatal testosterone, estradiol, and progesterone levels, as
assessed in amniotic fluid and maternal serum, were related to
sex differences and within-sex variations in gender-related
play behavior.
Since the literature suggests most clearly an effect of prenatal androgens on play behavior, we hypothesized that higher
levels of testosterone would be related to more masculine and less
feminine play behavior. Previous studies on children with CAH
indicate that these effects are clearly observed in girls (Berenbaum & Hines, 1992; Pasterski et al., 2005), but are absent in
boys (Berenbaum & Hines, 1992). In the maternal blood study,
Hines et al. (2002) reported the same pattern in a normative
population. Therefore, we predicted that this relationship would
be sex-specific, i.e., more clearly present in girls than boys. We
did not have clear predictions with respect to the behavioral
effects of variations in estradiol and progesterone.
Method
Participants
All participants were enrolled in a prospective longitudinal
project on the effects of prenatal sex hormones on gender
development. Participants were recruited from a consecutive
series of referrals, between January 1999 and August 2000, to
the Department of Obstetrics at the University Medical Centre
in Utrecht (UMCU) to undergo an amniocentesis because of
prenatal diagnostic screening. All participants lived in relative
close vicinity (30 km) of the UMCU. Amniotic fluid samples
were provided by 153 participants with a normal healthy
singleton pregnancy. Each participant gave informed consent
to the procedure and the UMCU Medical Ethical Committee
approved the study.
For the majority of the women (96%), the reason for
amniocentesis was a higher risk of age-related (36 years or
older) genetic changes and their implications. The others had
an amniocentesis because of their medical history (e.g., a
previous child with Down syndrome or radiation cancer
treatment) (3.3%) and one participant had a high result on the
triple test (indicating an increased risk for Down syndrome)
(0.7%). Seven participants reported that they did not become
pregnant spontaneously; n = 4 had ovulation induction of
which one also had donor insemination, n = 1 had artificial
insemination, and n = 2 had in vitro fertilization (IVF).
For 18 participants, data could not be collected at follow-up.
Three children could not be tested because of illness or pregnancy of their mothers. Two children were excluded when their
mother’s command of the Dutch language appeared to be
insufficient to complete questionnaires. One child was excluded
Arch Sex Behav (2009) 38:6–15
because of extreme prematurity (gestational period less than
35 weeks) at birth. Furthermore, we lost participants (n = 12)
because they moved abroad (n = 1) or were no longer interested to participate in the study (n = 11).
In total, 135 children were seen at the age of 13 months.
Two girls were so timid that behavioral observations were not
possible. The observations of three other children were not
reliable because of illness or fatigue and the play behavior of
four children could not be scored because of technical problems. In the end, the data of 126 children (63 boys and 63 girls)
were used for further analyses. The mean age of the children
was 56.4 weeks (SD = 1.2, range, 53.6–60.6). The mean age
of the mothers at amniocentesis was 37.5 years (SD = 2.1,
range, 28–45). The mean parental education score was 9.8
(SD = 3.1, range, 2–14). With respect to the presence of older
siblings, there were: no older brother (n = 69), one older
brother (n = 43), two or more older brothers (n = 10), no
older sister (n = 86), one older sister (n = 31), and two or
more older sisters (n = 5). None of these variables differed
significantly between boys and girls.
9
immediately following the amniocentesis. The hormonal
analyses were conducted as described above except for testosterone. In maternal serum, free testosterone, the biologically
active part of this sex hormone, was calculated using existing
procedures (Dunn, Nisula, & Rodbard, 1981). These equations
can be used to describe the relation between the bound and free
fraction of testosterone with other steroids and with several
binding proteins. We made calculations for a system of two
steroids (estradiol and testosterone) and two binding proteins
(albumin and SHBG). The value of albumin in maternal serum
was fixed at 43 g/l for measurements in the second trimester
and 35 g/l in the third trimester (Ashwood, 1994). Sex hormone binding globulin (SHBG) was measured by immunometric assay (SHBG Immulite, Euro/DPC, Gwynedd, UK);
interassay variation was 6.9% at a concentration of 93 nmol/l.
One girl with an extremely high (because of skewed distributions, a criterion of >4 SD was used) amnion testosterone
level (2.50 nmol/l) and whose mother had an extremely high
progesterone level (2,860.0 nmol/l) and one girl whose
mother had an extremely high plasma testosterone level
(42.40 nmol/l) were not included in the data analyses.
Measures
Sex Hormones in Amniotic Fluid
The amniotic fluid samples were collected between week 15.3
and 18.0 of pregnancy (M = 16.3, SD = .46). The length of
gestation was determined by the last menstrual period and/or
ultrasonic measurement of crown-rump length (CRL) (Daya,
1993). Because we had to adjust to the time schedule of the
clinic, it was not possible to standardize the hormone sampling
time completely; however, all samples were taken between
8:00 am and 13:00 pm. The material was stored at -30°C
until assayed. The lab employees who analyzed the samples
were masked to the behavioral data. Testosterone was determined by radioimmunoassay (RIA) after extraction with
diethyl ether (Van Landeghem et al., 1981). Interassay coefficient of variation was 8.8% at a testosterone level of
0.75 nmol/l and 9.4% at a level of 2.55 nmol/l. The measured
T level in amniotic fluid includes exclusively free T (FT) and
therefore needed no conversion. Estradiol (E2) (total) and
progesterone were determined using the Axsym of Abbott
(Abbott Park, IL). Sera were diluted with phosphate buffered
saline (0.01 M at pH 7.0, containing 0.5% bovine serum
albumin) (estradiol: second trimester serum 209; progesterone: 109). Interassay variation was 5.1% at 1,060 pmol/l for
estradiol and 5.0% at 18 nmol/l for progesterone.
Sex Hormones in Maternal Serum
We were able to collect serum in 115 mothers-to-be (57 boys,
58 girls). The maternal serum samples were collected
Play Observations
In a structured toy play session, nine different toys that previously had been classified by parents and non-parents as
masculine, feminine, or neutral were used (Campenni, 1999;
Servin et al., 1999). Each toy category was equally presented.
Neutral toys were a plastic friction dog, a wooden puzzle, and
a stacking pole with rings. Masculine toys were a trailer with
four cars, a garbage truck, and a set of three plastic pieces of
equipment. Feminine toys were a teapot with a cup, a soft doll
in a cradle with a blanket, and a doll with beauty set (brush,
comb, and mirror).
The toys were arranged in a standard order in a semicircle
(from left to right: tea set, dog, trailer, doll in cradle, rings, truck,
doll with beauty set, puzzle, and equipment). The mother was
asked to place the child in the center of the semicircle, at the
same distance from all toys, and then take a seat in the chair just
outside the semicircle (1.5 m). She was instructed to let the
child play on his or her own, but was allowed to give neutral
verbal reactions if the child specifically asked her attention.
When giving verbal reactions, she was asked to avoid naming
the objects and guiding the child in its actions. The child was
videotaped for 7 min, starting by the first touch of a toy. If play
behavior was not present for longer than 30 s (e.g., if the child
started to cry and sought comfort with his/her mother), this time
was added to the original 7 min. In this way, every child had
7 min real playing time. The play session took place at the
beginning of a more extensive testing session, after a short (5–
10 min) talk with the mother. The entire visit took approximately 1 h.
123
10
For each toy, we recorded the number of seconds the child
played with that particular toy. Each ‘‘play action’’ was scored
from the moment that physical contact started until the
physical contact stopped, unless there was still obvious
‘‘involvement’’ (e.g., pushing a car and crawling behind it).
‘‘Involvement’’ was defined as looking at, pointing at, and
moving behind the object. If the child played with several toys
simultaneously, physical contact time was scored for each toy
separately. Thus, the total playing time of a child (all time
spent with the different objects in sum) sometimes was longer
than the 7 min of play observation. For each toy category, the
total amount of time was counted and the percentage of the
total playing time was calculated, resulting in the variables ‘‘%
masculine play,’’ ‘‘% feminine play,’’ and ‘‘% neutral play.’’
Two participants with a deviant total playing time (>3 SD)
were excluded; one boy showed an extremely high (730 s) and
another boy an extremely low (36 s) total playing time.
The videotapes were scored by trained observers who were
masked to the hormonal values. The inter-rater reliability was
high. Kendall’s tau correlations ranged from .95 to 1.00 for
masculine play, from .94 to .99 for feminine play, and from .99
to 1.00 for neutral play.
Developmental Assessment
A home visit took place within a week after the play session.
During this visit, the Bayley Scales of Infant Development II
(BSID II) (Van der Meulen, Ruiter, Lutje Spelberg, & Smrkovsky, 2002) was administered to assess motor and mental
development.
Background Variables
Information was collected on several social and demographic
variables by means of a short questionnaire. The following
variables were used in the analyses: age of the mother at the
time of amniocentesis, parental educational level, and number
of older brothers and sisters. For each parent, the educational
level was scored on a 7-point scale, with a score of 1 indicating
‘‘no formal qualifications’’ to 7 representing ‘‘a university
degree.’’ The scores of both parents were combined.
Statistical Analyses
To standardize not normally distributed variables, a transformation was used. A square-root transformation was carried
out on the variables ‘‘% masculine play’’ and ‘‘% feminine
play.’’ The distribution of all prenatal sex hormones was
standardized by means of a logarithmic transformation. Student’s t tests were employed in statistical group comparisons
123
Arch Sex Behav (2009) 38:6–15
(one-tailed when comparing boys and girls on masculine and
feminine play, two-tailed in the other comparisons) and
Pearson correlation coefficients were calculated to map the
relationships among variables. Backward stepwise linear
regression analyses (removal criterion p =.10) were used to
examine the variance associated with relevant independent
and background variables that showed to be related (at
p < .10) to gender-related play. In the analysis including both
sexes, the dummy variable ‘‘sex’’ was coded 0 for boys and 1
for girls. Because sex and amniotic testosterone were so highly
correlated (r = -.78, df = 121, p < .001), it was not possible
to disentangle their separate relationships with play behavior
by means of regression analyses in the whole population
(multicollinearity). Therefore, for this hormone, we could
only analyze the data for boys and girls separately.
Results
Prenatal Sex Hormones
There were no significant sex differences in maternal serum
(see Table 1). In amniotic fluid, male fetuses had significantly
higher testosterone levels, t(120) = 13.20, p < .001 (d =
2.39), and female fetuses had significantly higher estradiol
levels, t(118) = -2.39, p < .05 (d = .43). No significant sex
difference was found with respect to amniotic progesterone
levels.
No significant relationship was found between maternal
age and the hormone levels, and the same applied for gestational age at sampling and maternal hormones. In male
fetuses, but not in female fetuses, there was a gestational age
effect with respect to the amniotic data. With the progression
of pregnancy, a significant decrease was found in testosterone
(r = -.33, df = 121, p < .05), progesterone (r = -.36, df =
121, p < .05), and estradiol (r = -.22, df = 119, p < .10).
To control for these effects, we used in further analyses the
standardized residual of the calculated regression lines instead
of the actual hormone levels.
Play Behavior
The total sample had a mean total playing time of 415 s
(SD = 87, range, 181–692). There was no significant sex
difference in the total time that the toys were handled.
Figure 1 shows the percentages of the total time spent with
the different toy categories for boys and girls. Boys (M = 40.96,
SEM = 3.59) spent a significantly higher percentage of time
playing with the masculine toys than girls (M = 27.10,
SEM = 2.87), t(120) = 2.92, p = .002 (d = .53). The percentage of time with feminine toys was significantly higher for
girls (M = 45.74, SEM = 3.43) than for boys (M = 36.62,
Arch Sex Behav (2009) 38:6–15
Table 1 Sex differences in
prenatal sex hormone levels
11
Hormones (nmol/l)
Male
M
Female
SD
n
M
p
SD
n
Maternal serum
Testosterone
Estradiol
Progesterone
6.78
3.91
56
7.15
3.81
56
ns
21.94
8.58
56
21.95
8.53
56
ns
176.36
49.90
56
173.71
54.06
56
ns
1.41
.36
61
.68
.28
61
<.001
.88
.26
61
1.01
.36
59
<.05
275.74
89.89
61
286.48
111.06
61
ns
Amniotic fluid
Testosterone
Estradiol
Progesterone
SEM = 3.56), t(120) = -1.92, p = .029 (d = .35). Boys and
girls did not differ significantly with respect to neutral play.
Gender-related Play and Non-hormonal Variables
In boys, no clear relationships were found between the age of
mother, parental education, the number of older sisters, and
mental and motor development, on the one hand, and masculine
and feminine play behavior, on the other, except for a marginally
significant positive relationship between parental educational
level and masculine play behavior. For feminine play, a marginally significant relationship was found for boys, but in the
opposite direction. In addition, the percentage of masculine play
behavior tended to be lower, and feminine play higher, with an
increase in the number of older brothers (see Table 2).
In girls, no significant relationships were found between
any of the background variables and gender-related play,
except for a significant negative relation between percentage
of feminine play and number of older sisters (see Table 2).
60
boys
girls
% of total playing time
50
*
40
**
30
20
10
0
Prenatal Hormones and Gender-related Play
No significant relations were found between maternal sex
hormones levels and masculine or feminine play behavior in
either boys or girls (see Table 2). With regard to the sex
hormones measured in amniotic fluid, a positive correlation
between progesterone and time spent with masculine toys
(r = .30, df = 121, p < .05) was observed in boys. No other
significant relationships with masculine or feminine play were
found in either boys or girls.
Regression Analysis Within the Sexes
Because there were no significant relationships between prenatal hormones and feminine play in boys or girls, and because
there was no relationship between prenatal hormone levels and
masculine play behavior in girls, we further explored the
relationship between hormones and masculine behavior in
boys only. First, the variables that were related to masculine
play (at a p < .10 level) were entered in the model simultaneously. These variables were progesterone in amniotic fluid,
parental educational level, and the number of older brothers.
Although amniotic testosterone was not significantly correlated with masculine play, we included it in the regression,
because amniotic testosterone and progesterone were positively related (r = .36, df = 121, p <.01). In the final model
(see Table 3), only progesterone in amniotic fluid significantly
and positively predicted masculine play. With respect to the
non-hormonal variables, masculine play was predicted by the
number of older brothers (less masculine play with more older
brothers) and parental educational level (less masculine play
with lower educational level).
Regression Analysis Including both Sexes
feminine
masculine
neutral
Fig. 1 Play with gender-specific toy categories ** p < .01, * p < .05
Using a similar procedure, we also entered sex, and the interactions between sex and amniotic progesterone and testosterone,
respectively, in the model (see Table 4). Again, progesterone
123
12
Arch Sex Behav (2009) 38:6–15
Table 2 Correlations between independent variables and gender-specific play categories
Masculine play
Feminine play
Boys
Girls
r
Boys
Girls
n
r
n
r
n
r
n
Hormones maternal serum
Testosterone
.09
56
-.01
56
-.17
56
.09
56
Estradiol
Progesterone
-.13
.12
56
56
.06
.22
56
56
.05
-.07
56
56
-.05
-.05
56
56
.11
61
.00
61
.01
61
.03
61
-.05
61
-.04
59
.04
61
.14
59
.30**
61
.07
61
-.19
61
.00
61
Mother’s age
.10
61
.03
61
-.11
61
.17
61
Parental education
.22*
61
.18
61
-.24*
61
-.20
61
Infant’s BSID II mental score
.02
60
.01
60
.03
60
-.01
60
Infant’s BSID II motor score
-.14
60
.05
60
.17
60
.11
60
No. of older brothers
-.23*
61
-.15
61
.23*
61
-.12
61
No. of older sisters
-.02
61
.21
61
61
-.31**
61
Hormones amniotic fluid
Testosterone
Estradiol
Progesterone
Background variables
-.04
** p < .05, * p < .10
Table 3 Regression model for predicting masculine play in 13-month-old boys (n = 61)
Predictor
B
SE B
Standardized b
Partial r
t
p
Progesterone in amniotic fluid
.70
.28
.30
.32
2.53
.014
Parental education
.19
.10
.24
.26
2.00
.050
-.71
.36
-.23
-.25
-1.96
.054
Older brother(s)
2
R = .194, F(4, 56) = 4.59, p = .006
Table 4 Regression model for predicting masculine play in all participants (n = 122) at age 13 months
Predictor
B
SE B
Standardized b
Partial r
t
p
Sex
-1.19
.39
-.26
-.27
-3.05
.003
Progesterone in amniotic fluid
.35
.20
.15
.16
1.75
.083
Parental education
.14
.06
.18
.20
2.15
.034
-.51
.27
-.16
-.17
-1.91
.058
Older brother(s)
2
R = .395, F(4, 117) = 5.40, p = .001
positively predicted masculine play, but now at a trend level.
Sex of child strongly predicted masculine play, as did parental
educational level. Finally, at a trend level, an inverse relation
was found between the number of older brothers and masculine
play.
Discussion
In the present study, we observed significantly higher amniotic
testosterone levels in male pregnancies, significantly higher
123
amniotic estradiol levels in female pregnancies, and no sex
difference in maternal plasma sex hormone levels. This is in line
with previous findings (Dawood & Saxena, 1977; Finegan et al.,
1989; Glass & Klein, 1980; Hines et al., 2002; Judd et al., 1976;
Nagamani et al., 1979; Robinson et al., 1977; Rodeck et al.,
1985). Furthermore, sex differences in toy preference were
clearly present at the age of 13 months. Boys spent significantly
more time playing with masculine toys than girls and girls
played significantly more with feminine toys than boys.
For both sexes, no significant relations were found between
amniotic testosterone or other hormones and masculine or
Arch Sex Behav (2009) 38:6–15
feminine play behavior, except for an unexpected positive
relationship between amniotic progesterone and masculine
play. This effect was clearly present in boys and was present at
a trend level in the total sample (girls and boys together).
We can think of several reasons for the absence of a significant relationship between amniotic testosterone and play
behavior. In our study, there are several methodological gaps
that may have led to insufficient experimental power. First, it
may be that one single sample of hormones does not represent
actual individual differences in fetal hormone exposure. Little is
known about circadian rhythms of sex hormones during pregnancy, but it has been reported that several hormones show
fluctuations within a day and across days, even in fetuses
(Séron-Ferré, Ducsay, & Valenzuela, 1993; Walsh, Ducsay, &
Novy, 1984). Furthermore, the relationship between testosterone measured in amniotic fluid and fetal blood is not
established, so it is still uncertain what the amniotic level really
represents.
Second, it may be that, at the age of 13 months, sex differences in play behavior were too small to study hormone–play
relationships in the currently used sample size. At 13 months of
age, we found clear sex differences in masculine and feminine
toy preference, but the effect sizes (Cohen, 1992) were small
(feminine play: d = .35) to moderate (masculine play:
d = .53). This is much smaller than what is usually reported in
studies in somewhat older pre-school children (e.g., d = 1.92
for masculine toys and d = 1.23 for feminine toys in 3-yearolds (Servin et al., 1999). However, other hormone studies did
find a relationship between amniotic testosterone and behaviors
that show sex differences of moderate effect size, i.e., eyecontact (d = .53) (Lutchmaya et al., 2002) and vocabulary size
(d = .67) (Lutchmaya et al., 2001). Therefore, we expected to
find at least significant results for masculine play.
Third, in a study on hormone–behavior relationships, an
instrument is needed that captures behavioral differences
between boys and girls, but that is also sensitive to within sex
differences. Despite the fact that we did find sex differences, it
may be that our method was not sensitive enough to measure
within sex variability. Although the mothers in our study were
instructed not to interfere, it may be that their presence has caused
some error.
The positive and unexpected relationship between amniotic progesterone and masculine play behavior in boys may
have reflected a Type I error, considering the multiple comparisons. Yet, in the Stanford Longitudinal Study (Jacklin,
Maccoby, & Doering, 1983; Jacklin, Maccoby, Doering, &
King, 1984; Marcus, Maccoby, Jacklin, & Doering, 1985),
which used umbilical cord blood assessments, some significant relationships were found between progesterone and traits
that can be labeled masculine. However, it is questionable
whether umbilical cord blood levels are a good index of
organizational effects of sex hormones (Cohen-Bendahan
et al., 2005) and little is known about the relationship between
13
progesterone levels from cord blood at birth and progesterone
levels in second trimester amniotic fluid.
Finally, some attention should be paid to the few potentially
relevant non-hormonal variables we incorporated in our study
to control for their possible effects. Although the design of the
study does not allow for definite conclusions, the results indicate that, already at 13 months of age, variables such as
parental education and the number of older brothers are related
to gender-related play behavior. In boys, there were indications
for a tendency of masculine play to be less frequent, and
feminine play to be more frequent, when the child had more
older brothers. Although having one older brother is associated
with more masculine gender role behavior (Rust et al., 2000),
studies that included families with more than one sibling
suggest that having two or more brothers is related to a
decrease in masculine behavior (Leventhal, 1970; SuttonSmith & Rosenberg, 1970). Rather than considering sibship as
a social factor some researchers consider it as a biological
factor. Blanchard (2001) proposed that each succeeding male
fetus leads to the production of antibodies that can pass through
the placental barrier, enter the fetal brain, and impede the
sexual differentiation of the brain in the male-typical direction.
Although both biological and social factors play a role in
gender role development (Ruble, Martin, & Berenbaum, 2006),
there is no real consensus as to if and how the different factors
interact and whether there is a specific point in time at which
social or cultural factors might, in fact, overrule potential
biological influences. Parental behavior may strengthen biologically based differences, augmenting small differences
present at 13 months. However, parents may also modify
individual preferences, and overrule biological predispositions,
for example in the case of very feminine boys. It is important to
follow up the participants of this study and establish whether the
studied relationships between prenatal hormones and genderrelated play behavior increase or change over time or only
appear later in development.
Acknowledgments We wish to thank Lieve Christiaens, Gerard
Visser, and the other members of the Department of Obstetrics for their
cooperation and Inge Maitimu and the employees of the Laboratory of
Endocrinology for performing the hormonal analyses. We wish to thank
the research assistants and students who helped to collect the behavioral
data. And above all, we are grateful to all mothers and children who
participated in the study. This research was funded by the Netherlands
Organization for Scientific Research NWO Grant No. 575-25-010.
References
Alexander, G. M. (2003) An evolutionary perspective of sex-typed toy
preferences: Pink, blue, and the brain. Archives of Sexual Behavior,
32, 7–14.
Alexander, G. M., & Hines, M. (2002). Sex differences in response to
children’s toys in nonhuman primates (Cercopithecus aethiops
sabaeus). Evolution and Human Behavior, 23, 467–479.
123
14
Ashwood, E. R. (1994). Clinical chemistry of pregnancy. In N.W. Tietz
(Ed.), Tietz textbook of clinical chemistry (p. 2112). Philadelphia:
Saunders.
Bailey, J. M., Bechtold, K. T., & Berenbaum, S. A. (2002). Who are
tomboys and why should we study them? Archives of Sexual
Behavior, 31, 333–341.
Berenbaum, S. A. (1998). How hormones affect behavioral and neural
development: Introduction to the special issue on ‘‘Gonadal
hormones and sex differences in behavior’’. Developmental Neuropsychology, 14, 175–196.
Berenbaum, S. A., Duck, S. C., & Bryk, K. (2000). Behavioral effects
of prenatal versus postnatal androgen excess in children with 21hydroxylase-deficient congenital adrenal hyperplasia. Journal of
Clinical Endocrinology and Metabolism, 85, 727–733.
Berenbaum, S. A., & Hines, M. (1992). Early androgens are related to
childhood sex-typed toy preferences. Psychological Science, 3,
203–206.
Blanchard, R. (2001). Fraternal birth order and the maternal immune
hypothesis of male homosexuality. Hormones and Behavior, 40,
105–114.
Caldera, Y. M., Huston, A. C., & O’Brien, M. (1989). Social interactions
and play patterns of parents and toddlers with feminine, masculine,
and neutral toys. Child Development, 60, 70–76.
Campenni, C. E. (1999). Gender stereotyping of children’s toys: A
comparison of parents and nonparents. Sex Roles, 40, 121–138.
Cohen, J. (1992). A power primer. Psychological Bulletin, 112, 155–159.
Cohen-Bendahan, C. C. C., Van de Beek, C., & Berenbaum, S. A.
(2005). Prenatal sex hormone effects on child and adult sex-typed
behavior: Methods and findings. Neuroscience and Biobehavioral
Reviews, 29, 353–384.
Collaer, M. L., & Hines, M. (1995). Human behavioral sex differences:
A role for gonadal hormones during early development? Psychological Bulletin, 118, 55–107.
Dawood, M. Y., & Saxena, B. B. (1977). Testosterone and dihydrotestosterone in maternal and cord blood and in amniotic fluid.
American Journal of Obstetrics and Gynecology, 129, 37–42.
Daya, S. (1993). Accuracy of gestational age estimation by means of
fetal crown-rump length measurement. American Journal of
Obstetrics and Gynecology, 168, 903–908.
Dunn, J. F., Nisula, B. C., & Rodbard, D. (1981). Transport of steroid
hormones: Binding of 21 endogenous steroids to both testosteronebinding globulin and corticosteroid-binding globulin in human
plasma. Journal of Clinical Endocrinology and Metabolism, 53,
58–68.
Ehrhardt, A. A., Meyer-Bahlburg, H. F. L., Feldman, J. F., & Ince, S. E.
(1984). Sex-dimorphic behavior in childhood subsequent to prenatal exposure to exogenous progestogens and estrogens. Archives of
Sexual Behavior, 13, 457–477.
Fagot, B. I. (1974). Sex differences in toddlers’ behavior and parental
reaction. Developmental Psychology, 10, 554–558.
Fagot, B. I. (1995). Parenting boys and girls. In M. H. Bornstein (Ed.),
Handbook of parenting: Vol. 1. Children and parenting (pp. 163–
183). Mahwah, NJ: Lawrence Erlbaum Associates.
Finegan, J. A., Bartleman, B., & Wong, P. Y. (1989). A window for the
study of prenatal sex hormone influences on postnatal development.
Journal of Genetic Psychology, 150, 101–112.
Finegan, J. A. K., Niccols, G. A., & Sitarenois, G. (1992). Relations
between testosterone levels and cognitive abilities at 4 years.
Developmental Psychology, 28, 1075–1089.
Fisher-Thompson, D. (1993). Adult toy purchases for children: Factors
affecting sex-typed toy selection. Journal of Applied Developmental Psychology, 14, 385–406.
Fitch, R. H., & Denenberg, V. H. (1998). A role for ovarian hormones in
sexual differentiation of the brain. Behavioral and Brain Sciences,
21, 311–352.
123
Arch Sex Behav (2009) 38:6–15
Forest, M. G., Ances, I. G., Tapper, A. J., & Migeon, C. J. (1971).
Percentage binding of testosterone, androstenedione and dehydroisoandrosterone in plasma at the time of delivery. Journal of
Clinical Endocrinology, 32, 417–425.
Gitau, R., Adams, D., Fisk, N. M., & Glover, V. (2005). Fetal plasma
testosterone correlates positively with cortisol. Archives of Disease
in Childhood, 90, 166–169.
Glass, A. R., & Klein, T. (1980). Changes in maternal serum total and free
androgen levels in early pregnancy: Lack of correlation with fetal sex.
American Journal of Obstetrics and Gynecology, 140, 656–660.
Gooren, L. J. G., & Kruijver, F. P. M. (2002). Androgens and male
behavior. Molecular and Cellular Endocrinology, 198, 31–40.
Goy, R. W., & McEwen, B. S. (1980). Sexual differentiation of the
brain. Cambridge, MA: MIT Press.
Grimshaw, G. M., Bryden, M. P., & Finegan, J. K. (1995). Relations
between prenatal testosterone and cerebral lateralization in
children. Neuropsychology, 9, 68–79.
Grimshaw, G. M., Sitarenios, G., & Finegan, J. A. K. (1995). Mental
rotation at 7 years: Relations with prenatal testosterone levels and
spatial play experience. Brain and Cognition, 29, 85–100.
Hines, M. (2004). Brain gender. Oxford: Oxford University Press.
Hines, M., Golombok, S., Rust, J., Johnston, K. J., & Golding, J.
(2002). Testosterone during pregnancy and gender role behavior
of preschool children: A longitudinal, population study. Child
Development, 73, 1678–1687.
Jacklin, C. N., Maccoby, E. E., & Doering, C. H. (1983). Neonatal sexsteroid hormones and timidity in 6–18-month-old boys and girls.
Developmental Psychobiology, 16, 163–168.
Jacklin, C. N., Maccoby, E. E., Doering, C. H., & King, D. R. (1984).
Neonatal sex-steroid hormones and muscular strength of boys and girls
in the first three years. Developmental Psychobiology, 17, 301–310.
Judd, H. L., Robinson, J. D., Young, P. E., & Jones, O. W. (1976).
Amniotic fluid testosterone levels in midpregnancy. Obstetrics and
Gynecology, 48, 690–692.
Kawata, M. (1995). Roles of steroid hormones and their receptors in
structural organization in the nervous system. Neuroscience
Research, 24, 1–46.
Knickmeyer, R., Baron-Cohen, S., Raggatt, P., & Taylor, K. (2005).
Foetal testosterone, social relationships, and restricted interests in
children. Journal of Child Psychology and Psychiatry, 46, 198–210.
Knickmeyer, R. C., Wheelwright, S., Taylor, K., Raggatt, P., Hackett,
G., & Baron-Cohen, S. (2005). Gender-typed play and amniotic
testosterone. Developmental Psychology, 41, 517–528.
Leventhal, G. S. (1970). Influence of brothers and sisters on sex-role
behavior. Journal of Personality and Social Psychology, 16, 452–465.
Lutchmaya, S., Baron-Cohen, S., & Raggatt, P. (2001). Fetal testosterone
and vocabulary size in 18- and 24-month-old infants. Infant
Behavior and Development, 24, 418–424.
Lutchmaya, S., Baron-Cohen, S., & Raggatt, P. (2002). Fetal testosterone
and eye contact in 12-month-old human infants. Infant Behavior
and Development, 25, 327–335.
Lytton, H., & Romney, D. M. (1991). Parents’ differential socialization
of boys and girls: A meta-analysis. Psychological Bulletin, 109,
267–296.
Marcus, J., Maccoby, E. E., Jacklin, C. N., & Doering, C. H. (1985).
Individual differences in mood in early childhood: Their relation
to gender and neonatal sex steroids. Developmental Psychobiology, 18, 327–340.
Meulenberg, P. M. M., & Hofman, J. A. (1991). Maternal testosterone
and fetal sex. Journal of Steroid Biochemistry and Molecular
Biology, 39, 51–54.
Morgan, B. L. (1998). A three generational study of tomboy behavior.
Sex Roles, 39, 787–800.
Nagamani, M., McDonough, P. G., Ellegood, J. O., & Mahesh, V. B.
(1979). Maternal and amniotic fluid steroids throughout human
Arch Sex Behav (2009) 38:6–15
pregnancy. American Journal of Obstetrics and Gynecology, 134,
674–680.
O’Brien, M., & Huston, A. (1985). Development of sex-typed play
behavior in toddlers. Developmental Psychology, 21, 866–871.
Pasterski, V. L., Geffner, M. E., Brain, C., Hindmarsh, P., Brook, C., &
Hines, M. (2005). Prenatal hormones and postnatal socialization
by parents as determinants of male-typical toy play in girls with
congenital adrenal hyperplasia. Child Development, 76, 264–278.
Rivarola, M. A., Forest, M. G., & Migeon, C. J. (1968). Testosterone,
androstenedione and dihydroepiandrosterone in plasma during
pregnancy and at delivery: Concentration and protein binding.
Journal of Clinical Endocrinology, 28, 34–40.
Robinson, J. D., Judd, H. L., Young, P. E., Jones, O. W., & Yen, S. S. C.
(1977). Amniotic fluid androgens and estrogens in midgestation.
Journal of Clinical Endocrinology and Metabolism, 45, 755–761.
Rodeck, C. H., Gill, D., Rosenberg, D. A., & Collins, W. P. (1985).
Testosterone levels in midtrimester maternal and fetal plasma and
amniotic fluid. Prenatal Diagnostics, 5, 175–181.
Ruble, D. N., Martin, C. L., & Berenbaum, S. A. (2006). Gender
development. In W. Damon & R. M. Lerner (Series Eds.) and N.
Eisenberg (Vol. Ed.), Handbook of child psychology (Sixth ed.).
Vol. 3: Social, emotional, and personality development (pp. 858–
932). New York: Wiley.
Rust, J., Golombok, S., Hines, M., Johnston, K., Golding, J., & The
ALSPAC Study Team. (2000). The role of brothers and sisters in
the gender development of preschool children. Journal of
Experimental Child Psychology, 77, 292–303.
Séron-Ferré, M., Ducsay, C. A., & Valenzuela, G. J. (1993). Circadian
rhythms during pregnancy. Endocrine Reviews, 14, 594–609.
Servin, A., Bohlin, G., & Berlin, L. (1999). Sex differences in 1-, 3-, and
5-year-olds’ toy choice in a structured play-session. Scandinavian
Journal of Psychology, 40, 43–48.
15
Servin, A., Nordenström, A., Larsson, A., & Bohlin, G. (2003). Prenatal
androgens and gender-typed behavior: A study of girls with mild
and severe forms of congenital adrenal hyperplasia. Developmental
Psychology, 39, 440–450.
Smail, P. J., Reyes, F. I., Winter, J. S. D., & Faiman, C. (1981). The fetal
hormone environment and its effect on the morphogenesis of the
genital system. In S. J. Kogan & E. S. E. Hafez (Eds.), Pediatric
andrology (pp. 9–19). The Hague, Netherlands: Martinus Nijhoff.
Sutton-Smith, B., & Rosenberg, B. G. (1970). The sibling. New York:
Holt, Rinehart & Winston.
Udry, J. R., Morris, N. M., & Kovenock, J. (1995). Androgen effects on
women’s gendered behavior. Journal of Biosocial Science, 27,
359–368.
Van de Beek, C., Thijssen, J. H. H., Cohen-Kettenis, P. T., Van Goozen,
H. M., & Buitelaar, J. K. (2004). Relationships between sex hormones
assessed in amniotic fluid, and maternal and umbilical cord blood:
What is the best source of information to investigate the effects of
fetal hormonal exposure? Hormones and Behavior, 46, 663–669.
Van der Meulen, B. F., Ruiter, S. A. J., Lutje Spelberg, H. C., &
Smrkovsky, M. (2002). Bayley Scales of Infant Development
(BSID) II. Dutch version. Lisse, Netherlands: Swets Test Publishers.
Van Landeghem, A. A. J., Poortman, J., Deshpande, N., Di Martino, L.,
Tarquini, A., & Thijssen, J. H. H. (1981). Plasma concentration
gradient of steroid hormones across human mammary tumors
in vivo. Journal of Steroid Biochemistry, 14, 741–747.
Walsh, S. W., Ducsay, C. A., & Novy, M. J. (1984). Circadian hormonal
interactions among the mother, fetus, and amniotic fluid. American
Journal of Obstetrics and Gynecology, 150, 745–753.
123