Subjects
We recruited two groups of healthy volunteers by posting advertisements and social networks including 26 nulliparous women (between the age of 24 and 32 years old, M = 26.68, SD = 1.84) and 25 men (between the age of 24 and 32 years old; M = 26.54, SD = 1.92). All subjects without any neurological, medical, psychiatric condition and no history of severe head trauma were screened for scanning after giving written consent to participate. Strictly right-handed individuals were assessed by the Chinese Hand Preference Questionnaire that was written according to the Edinburgh Handedness Inventory [27].
All procedures were approved by the Ethical Committee of East China Normal University Committee on Human Research (No. HR201508001). Each volunteer signed an informed consent form that was approved by the committee. All methods in our study were carried out in accordance with the principles outlined in the Declaration of Helsinki, including any relevant details.
Experimental materials and fMRI measurement
Before the experiment, we firstly selected a total of 120 color pictures of infant faces from the Chinese affective picture system [28]. These pictures included 40 happy infant faces, 40 neutral infant faces, and 40 sad infant faces. Every picture of size and background was unified to minimize any differences in stimuli’s physical characteristics. Then to exclude potential influence of other factors on brain activity, the pictures that were used in the scanner were selected within this database of 120 color pictures and rated by 29 adults (17 males, M = 25.10, SD = 1.89) on a 9-point Likert scale assessing arousal and valence. For arousal, the scale ranged from 1 (completely unaroused) to 9 (completely aroused), with a higher score meaning a higher arousal. For valence, the scale ranged from 1 (completely unhappy) to 5 (neutral) and to 9 (completely happy); The three groups of pictures differed significantly in each valence dimension [F = 91.13, p < 0.0001, Women, happy faces: M = 7.12, SD = 0.50; neutral faces: M = 5.14, SD = 0.35; sad faces: M = 2.63, SD = 0.60; Men, happy faces: M = 6.54, SD = 0.43; neutral faces: M = 5.12, SD = 0.29; sad faces: M = 2.84, SD = 0.45;]and arousal dimension [F = 16.80, p < 0.0001, Women, happy faces: M = 6.20, SD = 0.64; neutral faces: M = 3.50, SD = 0.89; sad faces: M = 6.60, SD = 0.80. Men, happy faces: M = 5.38, SD = 0.51; neutral faces: M = 2.87, SD = 0.68; sad faces: M = 6.09, SD = 0.60]. No significant differences were found between women and men in the arousal with FDR correction. They were recruited by public advertisement and participated in this behavioral experiment only. Each picture was presented for 3 s on a laptop randomly. Finally, we selected 60 stimuli including 20 happy, 20 neutral, and 20 sad faces from these 120 color pictures for the fMRI experiment. These pictures were balanced in arousal and valence. During the fMRI session, all subjects were presented with infant facial pictures in the scanner. Every picture was present with 2 s randomly, then followed by an average 4 s fixation cross (ranging from 2 s to 6 s). Every infant emotional face was presented twice, and the experimental paradigm was composed of 120 trials in total. This task has 363 volumes that last 12 min 6 s. All stimuli were presented using a SAMRTEC SA-9900 system (Shenzhen Sinorad Medical Electronics Inc., Shenzhen city, China). The SA-9900 system was used to provide synchronization between the stimuli presentation and the MRI scanner.
After the fMRI session, participants rated their arousal and valence while viewing a sample of 60 faces derived from the fMRI experiment stimuli. Each picture was projected on the screen for about 3 s in random order and participants subsequently rated its valence from “unhappy” to “happy” and its arousal from “calm” to “exciting,” using 9-point scales. The behavioral results were reported as follows: the valence and arousal of the internal consistency reliability coefficients examined in all infant emotional faces [valence: F (2, 59) = 125.53, p < 0.001, Cronbach’s Alpha = 0.89 and arousal: F (2, 59) = 58.55, p < 0.001, Cronbach's Alpha = 0.97]. Nulliparous women (happy: M = 7.59, SD = 0.86; neutral: M = 5.12, SD = 0.35; sad: M = 3.10, SD = 1.20) rated infant emotional faces more pleasant than men (happy: M = 7.26, SD = 0.75; neutral: M = 5.07, SD = 0.21; sad: M = 3.48, SD = 0.97), no significant differences were observed between women and men in feeling emotional infant faces with FDR correction. Nulliparous women (happy: M = 6.90, SD = 1.53; neutral: M = 3.11, SD = 2.01; sad: M = 6.68, SD = 1.50) also rated infant emotional faces more arousing than men (happy: M = 6.14, SD = 1.40; neutral: M = 2.93, SD = 1.65; sad: M = 6.36, SD = 1.22), no significant differences were found between women and men in the arousal with FDR correction.
All volunteers filled in the Interpersonal Reactivity Index (IRI) that used to assess participants’ empathic abilities [29, 30]. This IRI scale is a multi-dimensional assessment composed of 28 self-report items measuring four dimensions: The ‘empathic concern’ (EC) scale measures respondents’ prosocial feelings of warmth, compassion, and concern for others; The ‘personal distress’ (PD) scale measures self-oriented anxiety when observing others in distress; The ‘fantasy’ (FS) scale measures the tendency of the participant to identify with fictitious characters in books and movies; and the ‘perspective-taking’ (PT) scale assesses the tendency to take the psychological point of view of others. Studies reported that higher subscale scores are associated with higher empathic tendencies [30, 31]. The reliability of IRI analysis yielded high internal reliability coefficients with Cronbach’s Alpha = 0.86 in all subjects.
MRI image acquisition
The MRI scanning was performed on a Siemens 3.0 T Trio Tim MR system at the Shanghai Key Laboratory of Magnetic Resonance (East China Normal University, Shanghai, China). We used a 12-channel head coil for the whole brain scanning. Anatomical images were collected using a high-resolution T1-weighted 3-dimensional magnetization-prepared rapid-acquisition gradient-echo pulse sequence with the following acquisition parameters: repetition time (TR) = 2530 ms, echo time (TE) = 2.34 ms, flip angle = 7°, inversion time = 1100 ms, acquisition matrix = 256 × 256 mm2, field of view (FOV) = 256 mm, 192 slices. A T2*-weighted gradient-echo echo-planar-imaging sequence, which is sensitive to blood oxygen level-dependent contrast, was used to collect functional images, with the following parameters: TR = 2000 ms, TE = 30 ms, FOV = 220 × 220 mm2, acquisition matrix = 64 × 64, 33 slices, slice thickness = 3.5 mm, 25% gap. During the resting-state fMRI scan, we acquired 210 whole-brain volumes with the following parameters: TR = 2000 ms, TE = 30 ms, flip angle = 90°, slices = 33, transverse orientation, FOV = 220 × 220 mm2, matrix size = 64 × 64, slice thickness = 3.5 mm, and 25% distance factor. All subjects were instructed to rest, relax, and not think of anything, with their eyes closed.
MRI Data Analysis
Pre-processing of task-fMRI data
MRI data were analyzed with Statistical Parametric Mapping software (SPM12; http://www.fil.ion.ucl.ac.uk/spm/software/spm12) based on MATLAB 2015a. For data preprocessing: The preprocessing step contained slice timing correction, realignment, normalization to the Montreal Neurological Institute (MNI) space template, and spatial smooth was applied using a 4 mm Gaussian kernel. Slice timing correction was performed using the middle slice in time as reference. Spatial realignment was utilized to correct for head motion. The functional images were co-registered to the high-resolution T1-weighted images. The images were then spatially normalized to the MNI template (resolution of 3 mm × 3 mm × 3 mm) using the parameters obtained from segmentation. Finally, spatial smoothing with an 8 mm full-width half-maximum isotropic Gaussian kernel was performed on the functional images. Three subjects (one man and two women) were excluded because their translational head motion exceeded 2 mm or their rotational motion exceeded 2°. The remaining subjects entered the further statistical analysis.
Statistical analysis of task-fMRI data
After preprocessing, for each subject, images with an analytic design matrix were constructed, onsets and duration of each trial as epochs convolved with a hemodynamic response function used the general linear model in first-level statistical analysis. Six head movement parameters from realignment as regressions of no interest were also included. Three contrasts (happy, neutral, and sad) were designed. These images entered the second-level statistical analysis. To address our hypothesis of altered empathetic and reward processing in respond to infant emotional faces, we performed a 2 (group: nulliparous women vs. men) × 3 (condition: happy, neutral, and sad) ANOVA on whole-brain data using a flexible factorial model, with group as between-subject factor and emotional faces condition as within-subjects factor. Post hoc t-tests were conducted to further investigate potential interaction effects. The whole-brain significant results for functional imaging data were reported at the threshold of voxel level p < 0.001 and a cluster-size threshold of p < 0.05 corrected using family-wise-error (FWE).
Pre-processing of rs-fMRI data
The currents study also scanned rs-fMRI data. To examine whether differentially activated brain regions showing differences between nulliparous women and men in response to emotional infant faces acted in concert with other regions as a network, we performed FC analysis using rs-fMRI data. The resting-state data analysis was performed using DPABI [32]. The rs-fMRI data preprocessing applied in the research were as below: Original DICOM format was converted into NIFTI format; the first ten volumes of the functional images were discarded to allow for the magnetization to reach a steady state; the rest functional volumes were corrected with respect to slice timing and head motion correction by linear regression process, then images were realigned; structural images were co-registered to the average functional data, and normalized fMRI data were re-sliced with a resolution of 3 × 3 × 3 mm3; the processed data were smoothed with an 8-mm Gaussian kernel. The generated images were processed using linear trend removal and bandpass filter using 0.01 ~ 0.1 Hz. And several covariates of no interest were regressed from the data including head motion parameters, mean cerebrospinal fluid signal, mean white matter signal, and mean global signal. The residual time series of each subject was used to compute the resting-state FC.
FC analysis of rs-fMRI data
The preprocessed data was subjected to rs-FC analysis. Rs-FC maps for all subjects were obtained by calculating Pearson's correlation coefficient between ROIs and rest of the brain. First, the definition of ROIs: ROIs were defined from the significant differential brain regions in viewing infant emotional faces between women and men. The significant differential statistical parametric maps were from the above task-induced differential regions, these regions as seeds were explored to connect with the whole brain using FC method based on the rs-fMRI. The definition of ROIs used images calculator in SPM12. Then we computed the mean time series of the seeds and correlated these with the time series of other voxels in the whole brain to obtain FC maps. Finally, to improve the normality of the data distribution, FC maps were converted into z-score maps. Group effects were analyzed using the two-sample t-test in second-level statistical analysis. An initial threshold of p < 0.001 uncorrected was applied and results survived FWE correction at a cluster-level threshold of p < 0.05 were reported.
Assessment of IRI
All volunteers filled in the Interpersonal Reactivity Index (IRI) that used to assess a person’s empathy [33]. This IRI scale that is a persons’ multi-dimensional assessment of empathy consists of four sub-factors: cognitive empathy including Perspective Taking (PT) and Fantasy (FS); emotional empathy including Empathic Concern (EC) and Personal Distress (PD), respectively. Behavioral data of IRI are analyzed using SPSS 22 software with the independent sample t-test.