Hormonal and behavioral responses to an infant simulator in women with and without children

Abstract

We investigated the impact of maternal status on hormonal reactivity and behavioral responses to an infant simulator in 117 women (54 primiparous, 63 nulliparous). The amount of affectionate touch and motherese were analyzed as behavioral measures of caregiving. Saliva was collected before and 10 min after interaction with the infant simulator to analyze oxytocin, testosterone, cortisol, and estradiol levels. Nulliparous women also provided information about their fertility motivation. Linear mixed models indicated that greater use of affectionate touch was associated with lower overall testosterone levels. Cortisol decreased in response to the interaction in both groups. In the primiparous group, the amount of affectionate touch associated inversely with cortisol levels, whereas in the nulliparous group such association was not found. Oxytocin or estradiol reactivity to the simulator did not differ between the groups, nor were these hormones associated with behavior. Higher fertility motivation in nulliparous women was related to more motherese, and lower testosterone levels. Our results indicate that the simulator elicits hormonal reactivity both in mothers and nonmothers, but the patterns of associations between caregiving behavior and hormonal levels may be partially different. These results encourage using the infant simulator to explore hormonal processes related to the transition to parenthood.

Full text

Received: 8 March 2022 Revised: 1 August 2022 Accepted: 2 August 2022
DOI: 10.1002/dev.22321
RESEARCH ARTICLE
Hormonal and behavioral responses to an infant simulator in
women with and without children
Hanneli Sinisalo1Marian J. Bakermans-Kranenburg2Mikko J. Peltola1,3
1Human Information Processing Laboratory,
Faculty of Social Sciences, Psychology,
Tampere University, Tampere, Finland
2Faculty of Behavioural and Movement
Sciences, Educational and Family Studies,
Vrije Universiteit Amsterdam, Amsterdam,
The Netherlands
3Tampere Institute for Advanced Study,
Tampere University, Tampere, Finland
Correspondence
Hanneli Sinisalo, Human Information
Processing Laboratory, Faculty of Social
Sciences, Psychology, Tampere University,
Kanslerinrinne 1 P.O. Box 300, FI-33014
Tampere University, Finland.
Email: hanneli.sinisalo@tuni.fi
Funding information
Academy of Finland, Grant/AwardNumbers:
307657, 321424
Abstract
We investigated the impact of maternal status on hormonal reactivity and behavioral
responses to an infant simulator in 117 women (54 primiparous, 63 nulliparous). The
amount of affectionate touch and motherese were analyzed as behavioral measures
of caregiving. Saliva was collected before and 10 min after interaction with the infant
simulator to analyze oxytocin, testosterone, cortisol, and estradiol levels. Nulliparous
women also provided information about their fertility motivation. Linear mixed mod-
els indicated that greater use of affectionate touch was associated with lower overall
testosterone levels. Cortisol decreased in response to the interaction in both groups. In
the primiparous group, the amount of affectionate touch associated inversely with cor-
tisol levels, whereas in the nulliparous group such association was not found. Oxytocin
or estradiol reactivity to the simulator did not differ between the groups, nor were
these hormones associated with behavior. Higher fertility motivation in nulliparous
women was related to more motherese, and lower testosterone levels. Our results indi-
cate that the simulator elicits hormonal reactivity both in mothers and nonmothers,
but the patterns of associations between caregiving behavior and hormonal levels may
be partially different. These results encourage using the infant simulator to explore
hormonal processes related to the transition to parenthood.
KEYWORDS
caregiving behavior, cortisol, estradiol, fertility motivation, hormonal reactivity, oxytocin,
testosterone
1INTRODUCTION
The transition to parenthood in females is associated with major
biological and behavioral changes. In animals, the biological changes
associated with reproduction are well established (Brunton & Russell,
2008; Rilling, 2013), but in humans these changes are less well docu-
mented. Some earlier studies have indicated that gray matter volume
decreases during pregnancy in human mothers (Hoekzema et al., 2017;
Kim et al., 2010; Oatridge et al., 2002), and these changes are observed
in areas important for social cognition, empathy, and emotion regu-
lation (Adolphs, 2009;Kim,2016; Rocchetti et al., 2014), which are
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2022 The Authors. Developmental Psychobiology published by Wiley Periodicals LLC.
all essential for maternal behavior and sensitivity to infant signals. In
both women and men, parents and nonparents have also been shown
to differ in their brain activity in response to infant cues (Nishitani
et al., 2011; Peltola et al., 2014; Proverbio et al., 2006; Seifritz et al.,
2003; Witteman et al., 2019). In addition to changes in brain struc-
ture and functioning, hormonal changes during pregnancy have been
found in both mothers and fathers (Edelstein et al., 2015). Prenatal
changes in hormonal levels may be important for postnatal parenting,
as key parenting-related hormones such as oxytocin and testosterone
have been associated, for example, with attachment (Strathearn et al.,
2009), attraction toward infants (Fleming, Steiner, et al., 1997), and
Developmental Psychobiology. 2022;64:e22321. wileyonlinelibrary.com/journal/dev 1of17
https://doi.org/10.1002/dev.22321

2of17 SINISALO ET AL.
parental sensitivity (Feldman et al., 2007; Glynn et al., 2016). Cur-
rently, there are no studies comparing caretaking behavior between
new parents and nonparents or examining whether caretaking behav-
ior is similarly associated with hormone levels in these two groups.
Comparing parents and nonparents directly is important for under-
standing whether hormonal influences on caretaking behavior (or vice
versa) might change across the transition to parenthood. In this study,
we examine the behavioral correlates of oxytocin, testosterone, corti-
sol, and estradiol in primiparous and nulliparous women while they are
in interaction with an infant simulator.
1.1 Oxytocin
The neuropeptide oxytocin is produced and segregated in the hypotha-
lamus (Brunton & Russell, 2008) and it is essential for contractions
during labor (Blanks & Thornton, 2003) and lactation (Augustine et al.,
2018). Animal studies indicate that oxytocin is critical for the onset of
maternal behavior (Rilling & Young, 2014). For example, in rat dams,
oxytocin augments approach motivation toward pups through the
dopamine network, strengthening maternal behavior (Rilling, 2013).
In humans, plasma oxytocin levels may increase already when
women are in a relationship (Schneiderman et al., 2012). Levine
et al. (2007) observed that during pregnancy, some mothers dis-
played an increase in their plasma oxytocin levels, whereas others
showed decreasing or constant levels throughout pregnancy. Increas-
ing oxytocin levels during pregnancy also correlated with self-reported
prenatal bonding with the fetus (Levine et al., 2007). After pregnancy,
both maternal and paternal oxytocin levels increased similarly during
the first six postnatal months (Gordon et al., 2010). Higher oxytocin
levels in maternal saliva have been associated with more positive
behavior toward their 4- to 6-month-old infants (e.g., gaze toward
infant, positive affect, and infant-directed speech) and higher mother–
infant interaction synchrony (Feldman et al., 2011). Higher maternal
plasma oxytocin levels have also been associated with more affec-
tionate touch and eye contact when mothers engage in face-to-face
interaction with their infants (Feldman et al., 2012). Similarly, Gordon
et al. (2010) noticed that higher maternal plasma oxytocin levels were
associated with affectionate parenting behaviors (“motherese” vocal-
izations, expression of positive affect, and affectionate touch) when
assessed during the first six postpartum months. No studies, however,
have directly compared oxytocin levels in parents and nonparents or
investigated whether peripheral oxytocin levels are associated with
behavior toward infants also in nonparents. Investigating associa-
tions between oxytocin and maternal behavior in both mothers and
nonmothers is important for a better understanding of whether oxy-
tocin may be associated with caretaking behavior already before the
transition to parenthood.
1.2 Estradiol
Together with progesterone, the ovarian steroid estradiol prepares
the uterus for pregnancy and estradiol levels increase throughout
pregnancy, peaking just before birth (Edelstein et al., 2015; Fleming,
Ruble, et al., 1997; Glynn et al., 2016). Estradiol levels decrease rapidly
in the postnatal period in women (Fleming, Ruble, et al., 1997). In addi-
tion to reproductive functions, estradiol is associated with individual
differences in responses to emotional intimacy (Edelstein et al., 2010),
but the effects of estradiol on parent–infant interaction or mater-
nal behavior have been very little studied. One study observed that
mothers with lower estradiol increase during pregnancy showed more
sensitive parenting behavior 1-year postpartum (Glynn et al., 2016).
Relatedly, women with a lower prenatal estradiol increase were rated
as providing more spousal support by their partner (Edelstein et al.,
2017). The mechanisms underlying the somewhat paradoxical negative
relations between prenatal estradiol levels and postnatal parenting
outcomes are not clear, but it is possible that they are related to inter-
actions between estradiol and other hormones such as testosterone
(Bakermans-Kranenburg et al., 2022). However, the preliminary find-
ings are partially in line with studies on nonhuman primates, which have
associated higher estradiol levels during pregnancy with less optimal
maternal behavior toward offspring (Fite & French, 2000; French et al.,
2004).
1.3 Cortisol
Having an important role in fetal maturation, maternal cortisol lev-
els increase during pregnancy, remain elevated for the first postnatal
weeks, and then decline to their original levels both in plasma (Flem-
ing, Steiner, et al., 1997) and urine samples (Conde& Figueiredo, 2014).
Furthermore, cortisol levelsare higher in p rimiparous than multiparous
women (Bleker et al., 2017; Conde & Figueiredo, 2014) and this dif-
ference is partly mediated by pregnancy-specific distress in primiparas
who tend to experience more stress symptoms through pregnancy than
multiparas (Gillespie et al., 2018).
Higher salivary cortisol levels have been linked to higher sensibility
to the own infant’s body odor (Fleming, Steiner, et al., 1997) and higher
self-reported sympathy to crying infant stimuli in postpartum women
(Stallings et al., 2001). On the other hand, higher salivary cortisol levels,
measured both cross-sectionally and longitudinally, have been associ-
ated with lower maternal sensitivity (Finegood et al., 2016; Gonzalez
et al., 2012). In addition, lower salivary cortisol levels have been associ-
ated with less intrusive parenting behavior in mothers of 6-month-old
infants (Mills-Koonce et al., 2009). Thus, while elevated cortisol may
have a role in a more vigilant response to infant cues, it is negatively
associated with observed sensitive parental behavior, likely reflecting
the effects of stress on parenting.
1.4 Testosterone
Testosterone research has mainly focused on men, with results show-
ing decreasing testosterone levels during the transition to fatherhood.
However, testosterone has an important role in female parenting as
well, although the associations between testosterone and parenting

SINISALO ET AL.3of17
might be different in women. According to the Challenge hypothe-
sis, decreased testosterone levels facilitate investment in family life
and sensitive parenting behaviors in men (Archer, 2006; Gettler, et al.,
2011; Meijer et al., 2019). In line with these findings, higher testos-
terone levels have been associated with lower reproductiveambition in
young women (Deady et al., 2006). Testosterone levels change during
the prenatal period, but in a sexually dimorphic manner: testosterone
levels increase during pregnancy in mothers and decrease in fathers
(Edelstein et al., 2015). This suggests that results from male samples
cannot be generalized to women.
Postnatally, both fathers and mothers have lower testosterone lev-
els in general than nonparents (Barrett et al., 2013; Fleming et al., 2002;
Grebe et al., 2019; Meijer et al., 2019) and lower testosterone levels
are associated with better relationship quality in both men and women
(Edelstein et al., 2017). In line with the Challenge hypothesis, Weisman
et al. (2014) found that higher baseline testosterone is associated with
lower paternal sensitivity (gaze, touch, infant-directed speech). Simi-
larly, Fleming et al. (2002) observed that both fathers and nonfathers
with lower testosterone levels expressed a higher need to respond to
infant cries than men with higher testosterone levels. In women, higher
salivary testosterone levels have been associated with their motiva-
tion to view cute infant faces (Hahn et al., 2015). Furthermore, the
dual-hormone hypothesis suggests that the association of testosterone
with behavior depends on cortisol levels: higher cortisol levels may
inhibit the effects of testosterone on aggression or dominance (Mehta
& Josephs, 2010). The same effect can be found in men’s self-reported
empathy: when basal cortisol is low, high testosterone levels predict
lower empathy (Zilioli et al., 2015). However, the moderating effects of
cortisol on testosterone have so far been studied mainly in men (but
see Voorthuis et al., 2019).
1.5 Hormonal reactivity
Research on hormonal associations with parenting has largely focused
on hormonal baseline levels. Nonetheless, hormonal levels also show
short-term reactivity and the associations of such reactivity with
parenting behavior are an important research target. Trajectories of
hormonal baseline levels through pregnancy are well established but
whether short-term hormonal reactivity to infant stimuli changes
across the transition to parenthood is not known. Hormonal reactiv-
ity (measured from saliva or plasma) has been observed in response
to various triggers, such as exercise (Copeland et al., 2002; de Jong
et al., 2015), stressful situations (Cox et al., 2015; de Jong et al.,
2015), massage (Carter et al., 2007; Riem et al., 2017), and breast-
feeding (Grewen et al., 2010). Importantly, hormonal reactivity also
occurs in response to infant stimuli. For example, fathers’ salivary
cortisol levels decreased after interacting with their children (Gettler
et al., 2011) and listening to infant cry sounds has been associ-
ated with increases in oxytocin and cortisol levels in mothers (Swain
et al., 2011). In addition to responses to infant crying, parent–infant
touch is a significant aspect of parent–infant interaction. Skin-to-
skin contact has been found to increase oxytocin levels and decrease
cortisol levels both in infants and parents (Cong et al., 2015; Vittner
et al., 2018).
Hormonal reactivity has also been found to relate to parental behav-
ior. Kohlhoff et al. (2017) observed that in some mothers oxytocin
levels increased while in others oxytocin levels decreased or remained
constant when mothers and their 3- to 4-month-old infants were pre-
sented with the still-face paradigm. Increasing oxytocin levels were
associated with higher observed maternal sensitivity. Increased mater-
nal oxytocin levels after mother–infant interaction have also been
associated with more affectionate touch (Feldman et al., 2010)and
longer duration of mother-to-infant gaze (Kim et al., 2014). In addi-
tion, oxytocin responses to interaction with the infant have been found
to be larger in mothers with secure attachment representations as
measured with the Adult Attachment Interview (Strathearn et al.,
2009). In single women, attenuated estradiol reactivity to an emotion-
ally intimate parent–infant video was linked to avoidant attachment
style (Edelstein et al., 2012). In fathers, testosterone levels decreased
during the Strange Situation Procedure and the amount of decrease
predicted their sensitive parenting behavior when in interaction with
their own 12-month-old child (Kuo et al., 2016). Lotz et al. (2022)
found associations at trend level between fathers’ sensitivity and
both oxytocin reactivity and testosterone reactivity, with more sen-
sitive parenting behavior during a 10-min interaction with their own
2-month-old infant related to stronger increases in fathers’ oxytocin
and testosterone levels.
The impact of parental status on hormonal reactivity to infant
stimuli has not yet been studied. A few studies have used an infant sim-
ulator, which providesa validated and ethical method for studying both
parents and nonparents in a realistic caretaking situation (Bakermans-
Kranenburg et al., 2015; Voorthuis et al., 2013). The infant simulator
is a doll resembling a real infant in terms of weight, appearance, and
affective vocalizations. Testosterone levels in nulliparous women were
found to decrease after taking care of a crying infant simulator for
30 min (Voorthuis et al., 2019). A similar effect was observed in men
when they interacted with a crying but soothable infant simulator:
salivary testosterone levels decreased (van Anders et al., 2012). How-
ever, this finding did not replicate in an independent sample: instead
of a decrease, testosterone levels were found to stay constant when
men interacted with the crying simulator (van Anders et al., 2014).
Another study, on the other hand, observed increased testosterone
and cortisol levels in pregnant women following interaction with an
unsoothable infant simulator (Bos et al., 2018). Postnatally, when the
same participants interacted with their own infant, cortisol levels were
found to decrease. Thus, cortisol reactivity may be different before
and after birth or different to the mother’s own infant as compared
to an infant simulator. However, neither testosterone nor cortisol was
related to observed parental sensitivity in mothers, whereas in fathers
decreases both in testosterone and cortisol in response to interaction
were related to more sensitive parental behavior (Bos et al., 2018).
The associations between hormones (and their reactivity) and caretak-
ing behavior seem to be somewhat different in mothers and fathers,
and also in parents and nonparents, which makes it important to study
these groups separately.

4of17 SINISALO ET AL.
1.6 The present study
In this study, we investigated the impact of maternal status on salivary
hormonal reactivity and behavioral responses to an infant simulator.
Mothers and nonmothers have not been compared on these out-
comes and, therefore, the current study will provide important data
for understanding whether the associations between hormones and
behavior are independent of parental status. Primiparous and nulli-
parous women took part in a realistic caretaking situation with an
infant simulator that made both positive and negative sounds. Mater-
nal behavior was operationalized as the amount of affectionate touch
and motherese vocalizations (i.e., infant-directed speech) during the
caretaking situation. First, we investigated whether the two groups
differed in their hormonal reactivity toward the infant simulator. Due
to the lack of earlier studies comparing hormonal and behavioral
responses toward infants in parents and nonparents, we were cautious
in making strong directional hypotheses. Oxytocin reactivity has not
been investigated with an infant simulator, but based on research in
mothers with their own infants, oxytocin levels were expected to rise
at least in the primiparous group (Cong et al., 2015; Swain et al., 2011;
Vittner et al., 2018). Considering estradiol, we had a more exploratory
approach and did not make directional hypotheses. Based on the avail-
able evidence, we predicted that in nulliparous women testosterone
levels would decrease during the interaction (Voorthuis et al., 2019),
whereas in primiparous women testosterone levels were not expected
to show any reactivity,in line with the findings of Bos et al. (2018). Also,
according to Bos et al. (2018), one might expect differences in cortisol
responses in the two groups, with a decrease in primiparous women
and an increase in nulliparous women. However, as that study differed
in terms of study population and simulator paradigm from the cur-
rent study (i.e., pregnant women interacting with a persistently crying
simulator), we did not make strong directional hypotheses regard-
ing cortisol. Second, we investigated whether hormonal reactivity is
associated with behavior toward the infant simulator. We hypothe-
sized that positive oxytocin reactivity would be associated with greater
amount of affective touch and motherese vocalizations (Feldman et al.,
2010; Kim et al., 2014; Kohlhoff et al., 2017). Regarding estradiol,
testosterone, and cortisol, we did not have clear a priori hypotheses
due to insufficient earlier research. Based on earlier research on the
dual-hormone hypothesis (Mehta & Josephs, 2010), we also analyzed
whether the association between testosterone and maternal behav-
ior was dependent on baseline cortisol levels. Third, we investigated
whether the possible associations between hormones and behavior
were different in the two groups. Again, we adopted an exploratory
approach due to the nonexistent previous research comparing par-
ents and nonparents during interaction with an infant simulator.Finally,
in nulliparous women their attitudes and desires toward reproduc-
tion (i.e., fertility motivation; Brase & Brase, 2012; Deady et al., 2006)
might influence their interaction with the simulator or their hor-
monal reactivity. Thus, we investigated whether in nulliparous women
self-reported fertility motivation was associated with behavior and
hormonal levels as well as hormonal reactivity in response to the infant
simulator.
2METHODS
2.1 Participants
The participants were part of the TransParent project, which investi-
gates changes in processing infant cues during the transition to par-
enthood. The study protocol was reviewed by the Ethics Committee of
the Tampere Region. The participants were 22- to 37-year-old women
from Pirkanmaa area, Finland. Primiparous women were recruited by
invitation letters, which were sent based on contact information of
local primiparous families obtained from the Digital and Population
Data Services Agency. Nulliparous women were recruited through uni-
versity email lists. Inclusion criteria for all participants were the age
of 22–37 years, relationship duration longer than 6 months, living
with their partner, and sufficient skills in Finnish. In addition, nulli-
parous females were required not to have children (own or partner’s)
and primiparous women were required to have one approximately
6-month-old child (age at the time of the laboratory visit: M=7.19,
SD =1.48). Of the primiparous women, 55% were married and in the
nulliparous group the percent of married participants was 18%. The
majority (82%) of primiparous women were breastfeeding their infant
at the time of participation. Hormonal birth control (IUD, oral contra-
ceptives, ring, or capsule) was used by 63% of the nulliparous women
and by 20% of the primiparous women. In total, 117 participants
(54 primiparous, 63 nulliparous) visited the lab. Originally, we based
our targeted sample size of 120 participants on the key analyses of
the data collected during the laboratory visit, most of which had a
2×2 design. For such analyses, the targeted sample size had a power
of .80 (with an alpha level of .05) to detect main effects (i.e., group
differences) with an effect size of d=0.52 and larger, and two-way
interactions with an effect size of 𝜂2
p=.064 and larger. As a reward
for participation, the participants received one movie ticket and course
credit if necessary. Descriptive statistics are presented in Table 1.
2.2 Procedure
The participants were called before the laboratory visit. During the
call, menstrual cycle and hormonal contraceptive use were screened.
Whenever possible, the laboratory visit was scheduled in the luteal
phase of the cycle. The participants were asked not to eat or drink in a
full hour before the visit. Similarly, breastfeeding mothers were asked
to breastfeed their baby 1 h before the visit (time since breastfeed-
ing: Mhour =1.37, SD =1.04). These practices aimed at controlling the
effects of menstrual cycle (Engel, Klusmann, et al., 2019), contraceptive
use (Montoya & Bos, 2017;VanAndersetal.,2014), and breastfeed-
ing (White-Traut et al., 2009) on hormonal levels. Visits took place
between 12:00 p.m. and 6:00 p.m. to control for the diurnal variation
of hormone levels (Endendijk et al., 2016; Van Anders, Goldey & Bell,
2014). The whole laboratory session consisted of six different tasks and
lasted approximately 75–90 min. The majority of the tasks were com-
puterized tasks measuring physiological, attentional, and motivational
responses to infant stimuli. Those data will be reported elsewhere.

SINISALO ET AL.5of17
TAB L E 1 Descriptive statistics of the nulliparous and primiparous groups
Nulliparous Primiparous
nM(SD)nM(SD)t(df)χ2
Age 63 26.32 (3.44) 54 29.91 (2.96) 5.99 (115)***
Relationship duration in years 63 4.57 (3.53) 49 6.49 (3.33) 2.95 (110)**
Married 11 17.5% 27 55.1% 17.42***
Years of education 63 16.3 (1.96) 49 17 (2.34) 1.74 (110)
Menstrual cycle day 49 20.84 (10.53) 22 22.41 (7.22) 1.73 (72)
Time of day 63 13:38 (1:42) 54 14:17 (1:53) 1.864 (115)
Hormonal birth controla40 63.5% 11 20.4% 21.898***
Incomeb33.975***
<14,999 26 41.9% 2 4.1%
15,000–29,999 13 21.0% 510.2%
30,000–49,999 15 24.2% 15 30.6%
50,000–69,999 34.8% 15 30.6%
70,000–89,999 4 6.5% 10 20.4%
>90,000 1 2 4.1%
aUse of oral contraceptives, hormonal IUD, contraceptive implant, or contraceptive patch.
bAnnual household income in Euros.
**p<.01
***p<.001.
At the beginning of the laboratory visit, participants received infor-
mation about the study, signed an informed consent, and completed
a short questionnaire, which was followed by the first saliva sample
(T1). Samples were collected with a Salivette (Sarstedt, Nümbrecht,
Germany) polypropylene swab. Participants were asked to chew the
swab for 1 min and then insert the swab into a polyethylene container
without touching the swab with their hands. The swab was initially
stored at −20 to −30◦C before being transported in dry ice to a liq-
uid nitrogen freezer (−80◦C). The samples were analyzed in the Finnish
Institute of Occupational Health (Helsinki, Finland).
Before the infant simulator task, the participants performed a short
task and had two ECG electrodes attached to their chest. The elec-
trodes were attached to direct attention away from the video camera
and to make the physiological measurement feel more realistic to the
participants as they were told that the aim of the infant simulator
task was to “examine the physiological reactions caused by interac-
tion with the infant simulator.” The infant simulator was a lifelike
doll weighing about 5 kg (https://www.renates-puppenstube.de/en).
The doll had a small Bluetooth speaker inside. The sounds of the
infant simulator were vocalizations of real infants varying from full-
blown cry to delighted giggling sounds, which were collected from
the internet. Interaction with the infant simulator was videotaped
so that it was possible to observe the ongoing interaction from a
monitor in an adjacent room. During the infant simulator task, the
experimenter controlled the infant sounds with a Bluetooth-connected
laptop. The sounds were presented with VLC media player software.
The sequence of events was identical for all participants except for
the duration of the crying period. The infant simulator cried as long
as participants were changing the diaper and soothed right after the
task was done. The videos were recorded and used for behavioral
analysis.
The infant simulator was placed in a carrier in the testing room
before the participant’s arrival so that the participants were able to
see the simulator immediately upon entering the laboratory. Before
starting the infant simulator task, the experimenter took the simula-
tor to the adjacent room to turn on the speaker and connect it to the
laptop for the sound presentation. During this period, the participant
completed the 20-item Positive And Negative Affectivity Schedule
(PANAS; Watson et al., 1988) questionnaire that inquired about their
current affects. After this, the simulator was returned to the room in
the carrier. Participants were given the instruction to spend approxi-
mately 6 min (M=5.8 min, SD =0.56 min; Figure 1) with the simulator
and try to interact with it like they would interactwith a real baby. They
also received the instruction to try to soothe the baby if it started to get
fussy and that the baby would not calm down without a diaper change
if it started to cry very loudly. The testing room had a mat on the floor
with various toys, diapers, a changing pad, and wet wipes available.
Participants were then left alone with the simulator for approximately
6 min. The simulator made neutral to mildly positive vocalizations dur-
ing the first 2 min of the task. After 2 min, the simulator started to
whimper and soon after cry loudly. The simulator cried in total of 2 min
(M=1.86 min, SD =0.38 min). After the participant had changed the
diaper, the simulator began to make content vocalizations and eventu-
ally giggle for about 30 s. The purpose of this sequence of events was to
give the impression of a successful caregiving experience.
After the interaction with the simulator, the participants spent
10 min alone while filling in two questionnaires: one measuring self-
reported empathy (Interpersonal Reactivity Index; Davis, 1983)and

6of17 SINISALO ET AL.
FIGURE 1 Timeline of the study procedure (below) and timing of infant simulator sounds (above)
the PANAS questionnaire for the second time. After 10 min, the second
saliva sample (T2) was collected in the same way as the first sample. The
whole procedure is illustrated in Figure 1.
Within days after the laboratory visit, the participant received a
link to an online questionnaire, which included questions about back-
ground information such as education, income, and relationship status
and length. The questionnaire also included items assessing depressive
symptoms (CES-D; Radloff, 1977), anxiety (STAI; Bieling et al., 1998),
relationship satisfaction (CSI; Funk & Rogge, 2007), and reflective func-
tioning (RFQ; Fonagy et al., 2016). Primiparous women were also asked
about any pregnancy complications, the infant’s health, and maternal
postnatal attachment representations (MPAS; Condon, 2015). Nulli-
parous women were asked about their wishes of having children in the
future, experience of taking care of children, and their fertility motiva-
tion (Attitude Toward Babies Scale [ABS]; Brase & Brase, 2012). All of
the nulliparous and 94% of the primiparous participants completed the
online questionnaire.
2.3 Measures
2.3.1 Behavioral coding from the videos
In this study, we used a frequency-based method for analyzing behav-
ior, because it is feasible, objective, and suitable for evaluating behav-
ioral responses to predetermined stimuli like an infant simulator. The
videos were coded offline with Noldus Observer XT (version 11;
https://www.noldus.com/observer-xt) with a combination of continu-
ous and interval sampling. Using 5-s intervals, the prevailing behavior
of the participant was coded in three different categories that were
similar to the categories used in Feldman et al. (2011) and Gordon et al.
(2010). Adult vocalizations were coded with a 5-point scale: motherese
(high-pitched talk directed to the simulator), neutral talk (regular adult
talk directed to the simulator), talking to the self or the camera, singing,
and no vocalization. Motherese was considered as the best indicator of
sensitive behavior out of these types of vocalizations. Adult touch also
included five options: affectionate touch (hugging, soothing, cradling,
stroking, patting, that is, any touch that is meant to feel pleasant to
the child or is meant to soothe the child), stimulating touch (tickling,
“flying,” waving the simulator’s limbs), functional touch (e.g., lifting the
simulator’s leg while changing the diaper), hold (holding the simula-
tor without movement or further contact), and no touch. Affectionate
touch was considered as the most sensitive behavior in the adult touch
category. Adult affect was coded as either neutral, positive (smiling), or
negative (frowning). Positive affect was considered as the best indica-
tor of sensitive behavior in the adult affect category. Thus, each 5-s
interval received a score for each of the three categories.
Two coders, who were blind to the maternal status of the partici-
pants, double-coded 12 videos (10% of the sample), with an interrater
reliability (Cohen’s κ) of .76. Two behavioral measures were chosen for
the statistical analyses: the percentage of affectionate touch and the
percentage of motherese across all 5-s intervals. These two behavioral
measures were chosen because they reflect sensitive caregiving behav-
ior toward the simulator despite changes in the emotional state of the
infant simulator and because earlier findings have demonstrated their
association with oxytocin levels in mothers (Gordon et al., 2010). Adult
affect was excluded from the analysis because of insufficient data: too
often participants’ faces were not visible on the video, which prevented
reliable evaluation of the participants’ affect.
2.3.2 Oxytocin
Salivary oxytocin was analyzed with the Oxytocin ELISA kit (ENZO,
cat.no ADI-900-153A). Salivary samples were purified with solid-phase
extraction strata-X sorbent and 96-well plate with 60 mg wells (Phe-
nomenex 8E-S100-UGB). SPE columns were first revived with 1 ml of
methanol. Methanol residues were washed with 1 ml of water. A total
of 300 μl of 1.5% trichloroacetic acid (TFA)in water was added to 500 μl
of saliva, stirred, and centrifuged at 6000 ×gfor 10 min. Samples were
loaded into SPE columns, washed with 1.5 ml of 0.1% TFA, and eluted
with 1 ml of acetonitrile (0.1% TFA, 80:20). Eluent was evaporated
in vacuum centrifuge and samples were stored in a freezer (−20◦C)
until determination with the ENZO oxytocin ELISA kit according to the
assay procedure. The coefficients of variation percent of intraassay and
interassay of the method reported by the manufacturer were 12% and
16%, respectively. For oxytocin, we were able to analyze 64% of the
first saliva samples and 63% of the second samples. The rest of the sam-
ples were either too low in oxytocin or there was not enough saliva for
this assay.

SINISALO ET AL.7of17
2.3.3 Cortisol
Salivary cortisol was analyzed with chemiluminescence immunoassay
(LIA, IBL International, RE62011). Measuring range of the method is
0.43–88 nmol/L. The coefficients of variation percent of intraassay and
interassay of the method were 5% and 7%, respectively. The analysis
was successful for 94% of both of the two cortisol samples.
2.3.4 Testosterone
Salivary testosterone was analyzed with enzyme immunoassay for
the quantitative determination of free testosterone in saliva (EIA,
IBL International, RE52631). Measuring range of the method is
10–900 pg/ml. The coefficients of variation percent for intraassay and
interassay of the method were 6% and 9%, respectively. The analysis
was successful for 97% of both of the two testosterone samples.
2.3.5 Estradiol
Salivary estradiol was analyzed with luminescence immunoassay (IBL
International, RE62141). Measuring range is 0.3–64 pg/ml. The coef-
ficient of variation percent was 7.2%–13.3% for intraassay and 7.2%–
14.8% for interassay. Analysis was successful for 80% of the first
sample and 79% for the second sample. Estradiol was the last hormone
to be analyzed from the saliva samples and unfortunately in some cases
there was not enough saliva for the analysis (6% in the first sample and
9% in the second). Rest of the excluded estradiol samples were too low
in measuring range.
2.3.6 Positive and negative affects during the
interaction
Participants’ positive and negative affects before and after the inter-
action were inquired with the PANAS questionnaire (Positive And
Negative Affect Schedule; Watson et al., 1988), which consists of
20 words describing different emotions. Participants completed the
schedule before the interaction with the simulator and again imme-
diately after the interaction. Half of the words are positive (e.g.,
enthusiastic) and the other half negative (e.g., nervous). The participants
were asked to assess how much they were experiencing the affect in
question at that precise moment. The schedule is based on 5-point Lik-
ert scale (from 1 =very slightly or not at all to 5 =very much ).Sum scores
of all positive and negative ratings indicate the positive and negative
affect scales, respectively.
2.3.7 Fertility motivation
The fertility motivation of the nulliparous women was assessed with
the ABS (Brase & Brase, 2012). The Finnish version of the questionnaire
consisted of 16 items instead of the original 34 items and included eight
items of the Positive Exposure subscale and eight items of the Negative
Exposure subscale. The items of the Positive Exposure subscale included
positive experiences such as “Looking after other people’s babies makes
me want to have a baby of my own,” while the Negative Exposure subscale
included items such as “When I see an infant crying, I want to get as far
away from the noise as possible.” The items were coded in a 5-point Lik-
ert scale (ranging from 1 =Strongly disagree to 5 =Strongly agree,with
the Negative Exposure responses reversed) and an option for “Idon’t
know.” A fertility motivation composite was calculated as a mean of the
responses (Cronbach’s α=.95). Items responded with “I don’t know”
were replaced with the mean of the participant’s other values.
2.3.8 Covariates
Age of the participant, time of day, cycle day, relationship duration, and
years of education were initially considered as covariates based on ear-
lier research. For mothers, the age of the infant and the time from the
last breastfeeding were also included.
2.4 Statistical analyses
Video recordings were missing from three participants due to technical
problems, but these participants’ hormonal data were included in the
analysis. Two participants from the nulliparous group were excluded
altogether due to technical problems with the infant simulator.Outliers
in hormonal levels were examined and winsorized to 3 SDs (15 val-
ues in total). To achieve normality,oxytocin, estradiol, and testosterone
values were square root transformed, whereas cortisol was log trans-
formed. Hormonal reactivity scores were calculated as the difference
between second and first saliva samples divided by the value of the first
saliva sample. Reactivity scores were calculated from the winsorized
untransformed values and used for correlation analysis. Missing data
were estimated with multiple imputation implemented in IBM SPSS
Statistics 27. In total, five imputation rounds were performed, and the
pooled results are reported here. In total, 137 individual hormonal val-
ues were imputed (40 for T1 oxytocin, 41 for T2 oxytocin, 21 for T1
estradiol, 23 for T2 estradiol, five for each cortisol sample, and one for
each testosterone sample).
Associations between the hormonal, behavioral, and background
data were further investigated with Pearson correlation coefficients
separately within the two groups (Table 3). In addition, to explore
the influence of the categorically coded hormonal birth control use,
we compared hormonal levels between women who did and did not
use hormonal birth control separately in nulliparous and primiparous
women using independent samples t-tests. Two separate repeated-
measures ANOVAs were used to examine the change in participants’
positive and negative affects during the interaction task. Time (before
and after the interaction) was the within-subjects variable and parity
was the grouping variable.
As can be observed from Table 1, the groups differed in some back-
ground variables. Age and the use of hormonal birth control were
included as covariates in the main analysis. To answer the three main

8of17 SINISALO ET AL.
research questions, linear mixed models were conducted. One model
was built for each hormone making it in total of four models. The
−2-log likelihood ratio scale was examined as a determinant of model
fit. First, the repeated factor time (T1, T2) was added to the model,
followed by within-subjects covariates motherese, affectionate touch,
age, and a dummy variable representing the use of hormonal birth
control. Next, the between-subjects factor parity was added. For the
testosterone model, cortisol was also added as a covariate, to test
whether the association between testosterone and maternal behavior
was dependent on baseline cortisol level. Main effects of within- and
between-subjects variables were interpreted before adding any inter-
action terms to the models. Next, the interaction terms (parity ×time,
parity ×motherese, parity ×affectionate touch, time ×motherese,
time ×affectionate touch, and the interaction terms of cortisol and the
behavioral variables) were added. The final models are presented in the
results.
Finally, to explore the relation of fertility motivation to the behav-
ioral variables and hormonal reactivity within the nulliparous group,
Pearson correlation coefficients between the ABS questionnaire
scores and the behavioral and hormonal variables were calculated.
3RESULTS
3.1 Preliminary analyses
3.1.1 Descriptive statistics
As reported in Table 1, primiparous women were older than nulliparous
women (t(115) =5.99, p<.001, d=1.12), their households had higher
income (χ2=33.98, p<.001), and their relationships had lasted longer
(t(110) =−2.92, p=.004, d=0.56). Nulliparous women used hor-
monal birth control more often than primiparous women (χ2=21.90,
p<.001). The two groups did not differ in the phase of menstrual cycle
(t(72) =1.73, p=.088, d=0.43), years of education (t(110) =1.74,
p=.082, d=0.33), or the time of day (t(115) =1.86, p=.065, d=0.35).
The task duration did not differ between primiparous and nulli-
parous women (t(111) =1.37, p=.175, d=0.25), and the simulator
cried approximately the same time in both groups (t(111) =0.13,
p=.898, d=.02). Primiparous women expressed significantly more
motherese in interaction with the simulator (t(111) =4.18, p<.001,
d=0.79). There was no difference between primiparous and nulli-
parous women in the proportion of affectionate touch (t(111) =1.14,
p=.258, d=0.21). The descriptive statistics and sample sizes for
the winsorized untransformed values of the four hormones and the
behavioral variables are presented in Table 2.
Nulliparous women not using hormonal birth control had higher
baseline testosterone levels than nulliparous women who did use hor-
monal birth control (t(59) =2.53, p=.011, d=0.67). Other hormonal
baseline levels or reactivity did not differ as a function of hormonal
contraceptive use in either of the two groups. Within the primiparous
group, breastfeeding and non-breastfeeding women did not differ in
their baseline hormonal levels (Oxytocin: t(52) =−0.64, p=.524,
d=0.23; Estradiol: t(52) =1.31, p=.191, d=0.50; Testosterone:
t(52) =−0.53, p=.598, d=0.18; Cortisol: t(52) =0.90, p=.369,
d=0.32).
In nulliparous women, the covariates (age, educational years, rela-
tionship length, menstrual phase, duration of the simulator crying, or
time of day) did not correlate with any hormonal baselines or reac-
tivity, or with behavior toward the infant simulator. The correlation
coefficients between the study variables for nulliparous women are
presented in Table 3(below the diagonal).
In primiparous women, time from last breastfeeding was positively
correlated with baseline oxytocin (T1: r=.39, p=.01) and testosterone
reactivity (r=.46, p=.002). Time of day was inversely associated with
testosterone reactivity (r=–.31, p=.041). Educational years were
associated with the use of motherese (r=.34, p=.017). Other covari-
ates (relationship duration, menstrual phase, age, duration of simulator
crying, or own infant’s age) were not associated with any of the hor-
monal baseline levels, hormonal reactivity, or behavior toward the
infant simulator within the primiparous group. Correlation coefficients
for primiparous women are presented in Table 3(above the diagonal).
3.1.2 Positive and negative affect during the
interaction
A repeated-measures ANOVA showed a significant main effect of time
on positive affect (F(1, 114) =25.19, p<.001, 𝜂2
p=.18). Positive
affect increased from before to after the interaction with the simulator
(T1: M=29.15, T2: M=31.07). In addition, there was a significant main
effect of parity (F(1, 114) =5.78, p=.018, 𝜂2
p=.05), indicating that
primiparous women experienced more positive affect across time than
nulliparous women (T1: nulliparous M=27.92, primiparous M=30.54;
T2: nulliparous M=29.89, primiparous M=32.41). There was no inter-
action between parity and time (F(1, 114) =0.02, p=.899, 𝜂2
p=.00).
There was a main effect of time on negative affect (F(1, 114) =32.37,
p<.001, 𝜂2
p=.22): Negative affect decreased from before to after the
interaction (T1: M=15.17, T2: M=13.33). There was no main effect
of parity on negative affect (F(1, 114) =3.43, p=.067, 𝜂2
p=.03) nor
an interaction between parity and time (F(1, 114) =0.35, p=.558,
𝜂2
p=.00).
3.2 Hormonal reactivity and behavior towards
the infant simulator
The linear mixed models for all four hormones are presented in Table 4.
3.2.1 Oxytocin
Although mean values of the original data (Table 2) point to increased
oxytocin levels following the infant simulator in nulliparous females
and decreased levels in primiparous females, no significant main effects
of time or parity, or interactionsbetween parity and time were found in
the linear mixed model on the imputed dataset. Neither motherese nor

SINISALO ET AL.9of17
TAB L E 2 Descriptive statistics of the hormonal levels and behavioral measures separately for nulliparous and primiparous women
Nulliparous Primiparous
nM(SD) Min Max nM(SD) Min Max
T1
Oxytocin (pg/ml) 44 12.68 (11.71) 0.06 52.49 31 22.39 (32.76) 1.73 135.28
Estradiol (pg/ml) 47 2.51 (1.86) 0.05 7.26 47 2.38 (2.02) 0.09 9.49
Testosterone(pg/ml) 61 25.75 (15.64) 1.01 76.92 53 23.74 (11.85) 6.61 67.19
Cortisol (nmol/L) 57 4.07 (3.05) 1.18 16.72 53 5.33 (5.52) 0.67 27.97
T2
Oxytocin (pg/ml) 43 19.06 (29.19) 0.20 139.11 31 11.56 (6.43) 1.25 29.47
Estradiol (pg/ml) 48 2.25 (1.91) 0.01 7.76 44 2.42 (1.84) 0.01 8.28
Testosterone (pg/ml) 60 24.13 (13.76) 3.53 62.37 54 22.91 (13.53) 4.45 72.42
Cortisol (nmol/L) 57 3.73 (3.07) 0.90 14.74 53 4.28 (3.24) 0.32 15.18
Affectionate touch 59 37% 5% 75% 54 40% 7% 69%
Motherese*59 48% 0% 99% 54 72% 0% 99%
Note: Winsorized, untransformed, nonimputed data for the four hormones.
*p<.001.
TAB L E 3 Correlation coefficients between study variables
1 2 3 4 5 6 7 8 9 1011121314 15 16 171819
1 Oxytocin baseline – –.13 .17 –.17 .17 .40*.06 –.03 .10 –.13 –.04 .07 .09 .23 –.09 –.08 –.01 .39**
2Oxytocin reactivity .20 ––.06 .07 –.06 –.09 –.05 –.03 –.03 –.01 .00 .00 –.08 –.07 –.03 .04 .06 –.03
3 Estradiol baseline .21 –.15 – –.19 .04 –.02 .31*–.05 –.12 –.17 .02 .19 .10 .18 –.10 –.26 –.05 –.10
4Estradiol reactivity –.23 .22 –.36 –.04 –.00 –.07 .11 –.05 –.10 .00 –.04 –.15 –.12 .05 –.25 .00 –.05
5 Testosterone baseline .04 .25 .16 .05 – –.13 .22 –.01 –.19 –.36** .04 –.11 –.03 –.00 .23 .13 –.13 .08
6Testosterone reactivity –.06 –.07 –.06 .05 –.38** ––.04 –.13 .25 .01 –.08 .17 .15 .23 –.31*.03 .09 .46**
7 Cortisol baseline .12 –.24 .20 –.23 –.08 –.19 – –.22 –.04 –.33*–.07 –.04 –.00 .14 –.03 .04 –.02 .07
8Cortisol reactivity .05 –.08 –.17 .24 –.10 .21 –.07 ––.02 .04 .05 –.11 .19 –.24 .10 –.09 .04 –.08
9 Motherese –.21 .02 –.05 .00 –.30*.07 .18 .03 – .27*.18 .23 .28 .34*–.24 .15 –.06 .05
10 Affectionate touch –.18 –.08 .04 .07 –.17 .05 .13 .01 .28*–.22 .11 .15 .08 .08 .07 –.19 –.02
11 Duration of simulator crying .35 –.12 .19 –.08 .25 –.16 –.09 –.09 .02 .02 – .18 .00 .07 –.13 .20 –.14 –.13
12 Age .17 .05 –.06 –.04 .06 .19 .05 .08 .03 –.09 .06 –.43** .42** –.22 –.00 .29*.13
13 Relationship duration .23 .01 –.15 .09 .23 .03 –.05 .12 –.18 –.22 .08 .38**
–.36
*.04 .01 .12 .33*
14 Educational years .09 .17 .12 –.03 .09 –.07 –.00 .05 .22 –.14 .00 .57***.16 ––.00 .10 .02 .27
15 Time of day –.05 .16 –.02 .04 .11 .13 –.14 .08 –.12 –.09 –.11 .27*.14 .07 – –.15 –.10 –.13
16 Menstrual phase .06 .03 .13 .11 –.05 .11 –.04 .29 .05 .02 .23 –.32*–.09 –.28 .05 ––.17 –.11
17 Fertility motivation –.10 –.19 –.23 –.03 –.40*** .10 –.02 –.02 .47*** .15 –.07 –.17 –.18 –.08 –.19 .09 –
18 Age of infant –.35*
19 Time from breastfeeding –
Note: Data for nulliparous women are shown below the diagonal and for primiparous women above the diagonal.
*p<.05 (two-tailed)
**p<.01 (two-tailed)
***p<.001 (two-tailed).

10 of 17 SINISALO ET AL.
TAB L E 4 Linear mixed models
Models ICC Estimate SE 95% CI tp
Oxytocin .84
Intercept 2.76 1.49 [−0.18, 5.70] 1.85 .066
Time −0.04 0.14 [−0.32, 0.25] −0.25 .807
Parity −0.24 0.40 [−1.02, 0.54] −0.61 .543
Motherese −0.33 0.55 [−1.41, 0.76] −0.59 .555
Affectionate touch −1.83 1.08 [−3.98, 0.33] −1.69 .095
Age 0.08 0.05 [−0.02, 0.19] 1.52 .131
Hormonal birth controla−0.36 0.40 [−1.15, 0.44] −0.89 .375
Parity ×time −0.48 0.28 [−1.03, 0.08] −1.70 .091
Parity ×motherese 0.82 1.06 [−1.28, 2.91] 0.77 .443
Parity ×affectionate touch −1.49 2.13 [−5.69, 2.71] −0.70 .485
Time ×motherese −0.13 0.47 [−1.01, 0.80] −0.28 .784
Time ×affectionate touch 0.49 0.89 [−1.23, 2.26] 0.55 .586
Estradiol .68
Intercept 1.77 0.48 [0.83, 2.71] 3.68 .000
Time −0.5 0.05 [−0.15, 0.05] −0.93 .351
Parity −0.05 0.14 [−0.32, 0.22] −0.37 .712
Motherese −0.13 0.20 [−0.52, 0.26] −0.66 .511
Affectionate touch −0.14 0.34 [−0.81, 0.54] −0.40 .687
Age 0.00 0.02 [−0.03, 0.03] 0.04 .967
Hormonal birth control −0.21 0.12 [−0.45, 0.03] −1.73 .08
Parity ×time 0.17 0.13 [−0.09, 0.43] 1.35 .185
Parity ×motherese −0.05 0.35 [−0.73, 0.63] −0.14 .888
Parity ×affectionate touch −1.04 0.68 [−2.38, 0.30] −1.53 .127
Time ×motherese −0.16 0.21 [−0.60, 0.28] −0.74 .465
Time ×affectionate touch 0.06 0.32 [−0.57, 0.68] 0.18 .861
Testosterone .82
Intercept 5.81 1.14 [3.57, 8.04] 5.10 .000
Time −0.12 0.08 [−0.27, 0.03] −1.60 .110
Parity −0.11 0.31 [−0.71, 0.50] −0.36 .721
Motherese −0.60 0.42 [−1.43, 0.23] −1.42 .157
Affectionate touch −1.69 0.79 [−3.23, −0.15] −2.14 .032
Age 0.01 0.04 [−0.06, 0.09] 0.34 .732
Hormonal birth control −0.35 0.29 [−0.91, 0.21] −1.22 .222
Cortisol 0.08 0.44 [−0.79, 0.96] 0.18 .856
Parity ×time −0.15 0.16 [−0.47, 0.18] −0.88 .378
Parity ×motherese 0.97 0.83 [−0.65, 2.30] 1.17 .241
Parity ×affectionate touch −1.89 1.74 [−5.29, 1.51] −1.01 .276
Time ×motherese 0.53 0.26 [0.02, 1.04] 2.03 .043
Time ×affectionate touch −0.32 0.51 [−1.30, 0.65] −0.65 .514
Cortisol ×Parity 1.30 0.90 [−0.46, 2.60] −1.45 .148
Cortisol ×Motherese −0.14 1.37 [−2.82, 2.54] −0.10 .919
Cortisol ×Affectionate touch 2.17 2.79 [−3.38, 7.72] 0.78 .438
(Continues)

SINISALO ET AL.11 of 17
TAB L E 4 (Continued)
Models ICC Estimate SE 95% CI tp
Cortisol .21
Intercept 0.47 0.26 [−0.03, 0.98] 1.86 .063
Time −0.06 0.02 [−0.10, −0.02] −3.04 .002
Parity −0.05 0.07 [−0.09, 0.19] 0.67 .502
Motherese 0.09 0.10 [−0.11, 0.26] 0.81 .416
Affectionate touch −0.18 0.18 [−0.53, 0.17] −1.02 .310
Age 0.00 0.01 [−0.01, 0.02] 0.37 .710
Hormonal birth control 0.02 0.07 [−0.11, 0.15] 0.29 .775
Parity ×time −0.01 0.06 [−0.13, 0.13] −0.21 .835
Parity ×motherese −0.07 0.18 [−0.42, 0.29] −0.37 .712
Parity ×affectionate touch −0.88 0.38 [−1.63, −0.13] −2.32 .021
Time ×motherese 0.02 0.07 [−0.12, 0.15] 0.21 .832
Time ×affectionate touch 0.14 0.14 [−0.12, 0.41] 1.05 .292
Abbreviation: CI, confidence interval; ICC, intraclass correlation coefficient.
aDummyvariable(0=not using hormonal birth control, 1 =using hormonal birth control).
affectionate touch showed significant main effects or interaction with
parity or time.
3.2.2 Estradiol
For estradiol, there were no significant main effects of time or parity
nor were there interactions between parity and time. Neither moth-
erese nor affectionate touch had significant main effects or interaction
with parity or time.
3.2.3 Testosterone
For testosterone, there was a main effect of affectionate touch
(p=.032). More affectionate touch was associated with lower over-
all testosterone levels across the two measurement points. There were
no significant main effects of parity or motherese. Time showed a
significant interaction with motherese (p=.043). To illustrate this
interaction, standardized simple slopes were plotted for one standard
deviation below and above the mean for motherese (Figure 2). The
coefficients were in different directions signaling interaction. At low
levels of motherese, testosterone levels were decreasing (β=−0.28,
p=.346), whereas in high levels of motherese testosterone levels were
slightly increasing (β=0.153, p=.598).
Other interactions were not significant. To test for effects related to
the dual hormone hypothesis, baseline cortisol was also included in the
testosterone model. However, no interactions with cortisol emerged.
3.2.4 Cortisol
Time had a significant effect on cortisol (p=.002), with cortisol levels
showing an overall decrease from T1 to T2 (t(114) =2.84, p=.005,
d=0.28) as can be observed from Figure 3. There was also a sig-
nificant interaction between parity and affectionate touch (p=.021).
We examined this interaction with additional linear mixed models con-
ducted separately for both groups. For primiparous women, we found
a significant main effect of affectionate touch on cortisol (p=.035),
as more affectionate touch was associated with lower overall cortisol
levels across the two measurement points (Figure 4). For nulliparous
women, there was no significant main effect of affectionate touch on
cortisol levels (p=.312).
3.3 Fertility motivation and its associations with
behavior and hormones in nulliparous women
In nulliparous women, fertility motivation scores were positively asso-
ciated with the amount of motherese used with the infant simulator
(r=.47, p=.001) but not with affectionate touch. Fertility motiva-
tion was inversely correlated with baseline testosterone (T1: r=–.40,
p=.001). The correlation coefficients are presented in Table 3(below
the diagonal).
4DISCUSSION
The primary aims of the present study were to explore potential dif-
ferences in salivary hormonal reactivity and their associations with
behavioral responses in a simulated caretaking situation in primiparous
and nulliparous females. The participants spent 6 min taking care of
an infant simulator in a laboratory setting. The simulator made sounds
ranging from crying to laughter mimicking a real infant and the par-
ticipants were instructed to interact with the simulator as with a real
infant. Salivary levels of oxytocin, cortisol, estradiol, and testosterone
were measured before and after the situation to examine hormonal

12 of 17 SINISALO ET AL.
FIGURE 2 Regression lines representing the association between time and testosterone levels at low and high levels of motherese (±1SD)
FIGURE 3 Cortisol reactivity to the infant simulator in primiparous and nulliparous women
changes in response to exposure to the infant simulator, and indicators
of sensitive caregiving behavior (affectionate touch and motherese
vocalizations) were coded.
Cortisol levels decreased in response to the infant simulator in the
whole sample. In line with the PANAS scores showing increased pos-
itive affect and decreased negative affect following interaction with
the infant simulator, decreased cortisol levels suggest that the situa-
tion was not stress-evoking and nulliparous and primiparous women
were similar in their cortisol reactivity toward the infant simulator.
Decreasing cortisol levels have also been observed in mothers when
they take care of their own infant (Bos et al., 2018). Thus, the infant
simulator appears to produce similar cortisol reactivity compared to
interaction with a real infant. The decreasing cortisol levels in this study
were opposite to earlier results with pregnant women in Bos et al.
(2018), which suggests that during pregnancy cortisol reactivity may
differ from other life situations.
We also observed an interaction between the amount of affection-
ate touch and parity in overall cortisol levels. In primiparous women,
lower cortisol levels across the measurement points were associated
with more affectionate touch, whereas in nulliparous women no asso-
ciation between cortisol levels and affectionate touch was found. The
same difference was evident from the correlation coefficients (Table 3).

SINISALO ET AL.13 of 17
FIGURE 4 Correlation between overall cortisol levels and affectionate touch in primiparous women
This finding is novel and suggests that the association between cortisol
and affectionate touch might be different in primiparous and nulli-
parous women. Earlier studies have not compared primiparous and
nulliparous women in this regard, but our finding of an inverse rela-
tion between cortisol levels and affectionate touch in mothers is in
line with earlier research suggesting that lower baseline cortisol lev-
els are associated with higher maternal sensitivity (Finegood et al.,
2016; Gonzalez et al., 2012) and less intrusive parenting behavior
(Mills-Koonce et al., 2009). In addition, mothers’ sensitivity toward
an infant simulator has been observed to correlate with their sensi-
tivity toward their own infant (Bakermans-Kranenburg et al., 2015).
Together with the current study, those findings support the use of
the infant simulator as a valid method for investigating individual dif-
ferences in caretaking behavior and suggest that mothers differ from
nonmothers regarding the association between cortisol and caretaking
behavior also during naturalistic caretaking situations. In the current
study, mothers also used more motherese with the simulator than non-
mothers, suggesting that they were behaving with the simulator as with
their own infant. However, we did not control for the reported seri-
ousness or reality value (see Bos et al., 2018; Voorthuis et al., 2013)
of the interaction with the simulator, which is necessary in the future
studies.
There was a negative association between testosterone levels and
the amount of affectionate touch when taking care of the simulator.
This is in line with the earlier research with male samples (Bos et al.,
2018; Meijer et al., 2019; Weisman et al., 2014) and thus indicates that
testosterone might have similar associations with caretaking behav-
ior in females and males. We also observed an interaction between
time and motherese for testosterone levels: at low levels of moth-
erese, testosterone levels decreased in response to the interaction
with the infant doll, whereas at high levels of motherese testosterone
levels increased during the interaction with the doll. However, when
studying mothers, Bos et al. (2018) did not find any associations
between prenatal testosterone levels and caretaking behavior toward
an infant simulator, nor did they find any associations between postna-
tal testosterone levels and caretaking behavior with their own infant.
Comparisons between the current study and Bos et al. (2018) should
be made cautiously, however, as different interactive behaviors were
observed in these studies. While Bos et al. (2018) used an overall index
of maternal sensitivity, in our study we focused on more focal aspects
of behavior (motherese and affectionate touch). It is therefore possible
that the differences in the results of these studies may depend on the
different coding schemes. In the future, it will be important to evaluate
the feasibility of different coding schemes within the same sample.
In this study, primiparous and nulliparous women did not show dif-
ferent associations between testosterone and caretaking behavior in
the main analysis. In addition, we found no evidence of testosterone
reactivity to the infant simulator (i.e., a main effect of time) in the
full sample nor a difference in reactivity between primiparous and
nulliparous women. In earlier studies, testosterone levels have been
found to increase in pregnant women taking care of an infant simula-
tor(Bosetal.,2018), whereas in nulliparous women testosterone levels
have been found to decrease (Voorthuis et al., 2019). Thus, our find-
ings differ especially from those of Voorthuis et al. (2019) regarding
the nulliparous group. However, the infant simulator paradigm in our
study differs in important ways from those of earlier studies that have
included longer periods of crying and the lack of possibilities to sooth
the infant simulator. This may have triggered stronger testosterone
reactivity. Furthermore, we did not find cortisol to moderate the asso-
ciation between testosterone and behavior. One reason for this might

14 of 17 SINISALO ET AL.
be due to the all-female sample in our study. Earlier human and animal
studies have had either mixed-sex or all-male samples and it is possi-
ble that the moderating effect of cortisol on testosterone levels is more
evident in males than females.
The two groups did not show oxytocin reactivity toward the infant
simulator, nor did they differ in their oxytocin reactivity toward the
simulator. This was contradictory to our expectations and results from
studies investigating parental oxytocin reactivity in response to inter-
action with their infant (e.g., Feldman et al., 2010). However, in our
study, the amount of oxytocin was below the manufacturer’s limit in
48% of the sample and 36% of the saliva samples were unanalyzable
due to the low levels of oxytocin or insufficient amount of saliva, which
means that oxytocin data were based on imputed values to a greater
extent than was the case for other hormones. Therefore, the oxy-
tocin results should be evaluated with caution. The wide variability of
reported oxytocin levels across the literature has resulted in criticism
toward measuring oxytocin from saliva (Horvat-Gordon, et al., 2005;
McCullough et al., 2013). Many of the previous studies that reported
highly variable oxytocin levels were missing the extraction step of the
analysis. In this study, the oxytocin samples were extracted before
the assay, which, as a downside, partially explains the attrition in the
oxytocin data. Furthermore, similar to earlier studies, in our sample
baseline oxytocin levels were correlated with the time since the last
breastfeeding in mothers. This indicates that oxytocin levels as mea-
sured in the present study reflect true variation as in earlier studies
oxytocin levels have been found to start to increase after breastfeed-
ing reaching their peak just before the next feeding (Carter et al., 2007;
de Jong et al., 2015; White-Traut et al., 2009). It is noteworthy that
a considerable proportion of the literature documenting associations
between peripheral oxytocin and parenting behaviors is based on data
from a single laboratory (see Grumi et al., 2021). Therefore, it is vital
to replicate and extend such associations with greater diversity of
samples and methods.
Compared to other hormones in this study, there is very little
research on the relation of estradiol to caretaking in humans. In our
study, estradiol levels did not change in response to interaction with
the simulator, nor were they associated with caretaking behavior. The
few earlier studies linking estradiol levels to parenting or relation-
ship outcomes (Edelstein et al., 2017; Glynn et al., 2016) have studied
estradiol levels during pregnancy. As estradiol levels increase during
pregnancy and decrease rapidly after giving birth (Fleming, Ruble, et al.,
1997), associations between estradiol and caretaking behavior may be
more prominent during the prenatal period. In addition, associations
between estradiol and behavior might depend on individual proges-
terone or testosterone levels, which have been found to be important
in earlier studies. For example, smaller decline in the estradiol to pro-
gesterone ratio during pregnancy has been associated with higher
postpartum feelings of attachment toward the infant (Fleming, Ruble,
et al., 1997), and in fathers high testosterone levels combined with high,
but not low, estradiol levels have been associated with lower sensitiv-
ity (Bakermans-Kranenburg et al., 2022). In the future, it is relevant to
also measure progesterone, which might have a moderating effect on
estradiol reactivity.
In nulliparous women, higher fertility motivation was associated
with more motherese directed to the simulator. This finding is novel
and indicates that women’s positive feelings toward infants and moti-
vation to become a mother affect their caretaking behavior in a positive
way. In addition to behavior, fertility motivation was negatively asso-
ciated with testosterone levels. In line with the Challenge hypothesis
(Archer, 2006), the inverse relation between fertility motivation and
testosterone may suggest that, similarly to men, preparation to parent-
hood is associated with declining testosterone levels also in women.
Another possible explanation for the negative association between
testosterone and fertility motivation could be that women who have
lower testosterone levels in general have more positive views on
babies. This would also be in line with earlier results showing higher
testosterone levels to be associated with lower self-rated reproduc-
tive ambition (Deady et al., 2006). Fertility motivation or “baby fever,”
although being a popular subject on the media, has not yet been stud-
ied extensively. It is unclear whether fertility motivation predicts future
pregnancy in women. In addition, the potential associations between
fertility motivation and hormones and caretaking behavior in men are
an important target for future studies.
Together with earlier studies using the infant simulator (Bos et al.,
2018; Voorthuis et al., 2019), this study supports the use of the infant
simulator in comparing mothers and nonmothers: the infant simulator
elicited hormonal reactivity in both mothers and nonmothers, and the
two groups showed similar and partially different patterns of associa-
tions between hormonal levels and caregiving behavior. However, our
results are preliminary at best and require replication in independent
samples and greater variability of caregiving behaviors in the future. In
addition, it remains unclear when during the transition to parenthood
the potential differences in hormonal reactivity and caretaking behav-
ior begin to emerge. Longitudinal research designs would be important
to determine whether the differential responses emerge in mothers
due to the biological changes associated with pregnancy or whether
they are induced by caretaking experiences with their infants. Compar-
ing biological and adoptive parents in a simulated caretaking situation
might reveal an answer to this question. In addition, the impact of
hormones on different aspects of caretaking behavior may be indirect
and operate through motivational processes that may affect parental
behavior. For example, oxytocin has been linked to approach motiva-
tion (MacDonald & MacDonald, 2010; Soriano et al., 2020) and activity
of the reward system of the brain (MacDonald & MacDonald, 2010).
The heightened motivation toward babies could thus promote infant-
oriented behavior such as the use of motherese. In future studies, it
will be important to investigate the role of motivation toward babies
for parental behavior in mothers and nulliparous women in greater
detail.
ACKNOWLEDGMENTS
The authors would like to thank Prof. Anna Rotkirch for providing the
Finnish version of the fertility motivation questionnaire. This research
was supported by grants from the Academy of Finland (#307657 and
#321424).

SINISALO ET AL.15 of 17
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
Data are available on Open Science Framework: https://osf.io/8rh26/.
ORCID
Hanneli Sinisalo https://orcid.org/0000- 0003- 4987-1301
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How to cite this article: Sinisalo, H., Bakermans-Kranenburg,
M. J., & Peltola, M. J. (2022). Hormonal and behavioral
responses to an infant simulator in women with and without
children. Developmental Psychobiology,64, e22321.
https://doi.org/10.1002/dev.22321