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From July to September 2008, a longitudinal study of pregnant women and their subsequent infants (i.e., dyads) was performed. The women were recruited from the Brazilian National System of Public Health in Pelotas, a southern Brazilian city. The subjects underwent prenatal follow-up, and the inclusion criteria included the following specifications: more than 18 years old, living in the urban area, not taking antidepressants or mood regulators and capable of understanding and completing the neuropsychiatric questionnaires in the postpartum period. Between 60 to 90 days after delivery, the women were re-interviewed at home and provided blood for NGF, BDNF, and IL-6 determination and saliva for cortisol analyses. Cortisol was measured using salivary levels in both mothers and infants. In the 4th month of life, infants were evaluated for neurobiological - motor development. All women answered a confidential self-administered questionnaire that included questions about socioeconomic conditions (according to the Economic Classification for Brazil of the Brazilian Association of Population Survey Companies, in which the highest-income level is “A” and the lowest “E”), age, previous psychiatric treatment, use of psychoactive drugs in the past, previous suicide attempts, smoking habits, alcohol consumption, living with the father and the type of delivery. PPADs (depression, hypomanic/manic and mixed episode) and anxiety disorders were assessed using the Mini International Neuropsychiatric Interview (MINI Portuguese - version 5.0 Plus) [29]. MINI is a clinical structured interview that is compatible with DSM-IV criteria. It establishes a dichotomic variable for the presence or absence of PPAD. Stressful life events (SLEs) were assessed by means of six questions obtained from life events and coping scales [30]. The women were questioned in the postpartum period about the following issues during the gestational period: the death of someone in the family, grave sickness, a change of address, unemployment, disengagement and the risk of abortion. The Alberta Infant Motor Scale (AIMS) was used to evaluate the infants' neurobiological -motor development [31]. The AIMS, which is an observational assessment scale, is designed to measure gross motor maturation in infants from birth through independent walking (18 months). Based on the literature, 58 items were generated and organized into four positions: prone, supine, sitting and standing. Each item describes three aspects of motor performance: weight-bearing, posture and antigravity movements. Each participant's raw score was obtained by adding the scores of each scale. The raw scores and the ages of the infants were displayed in a graph in order to identify the percentile of infant neurobiological - motor development. In this study, we used the AIMS score for analysis. Three experienced physiotherapists who were blinded to the outcomes applied the AIMS testing. A concordance analysis was performed at the beginning and in the middle of the measurements. Peripheral venous blood samples (5 ml) were collected in anticoagulant-free tubes (vacutainer system) from all subjects between 08:00 and 10:00 a.m. The samples were immediately centrifuged at 5,000×g for 10 min, and the serum was stored at −80°C until analysis. Serum levels of NGF, BDNF and IL-6 were measured using a commercially available enzyme immunoassay kit. The amounts of BDNF, NGF and IL-6 were determined by measuring the absorbance at 450 nm with a SpectraMax M5 spectrophotometer. All samples and standards were measured in duplicate, and the coefficient of variation was less than 5%. The serum NGF, BDNF and IL-6 levels are expressed as ng/ml. Saliva samples (1 ml) were collected in sterile microtubes from all subjects between 08:00 and 10:00 a.m. The samples were immediately centrifuged at 5,000×g for 10 min, and the supernatant was stored at −80°C until analysis. Salivary cortisol was measured in duplicate samples with a commercially available High Sensitivity Salivary Cortisol enzyme immunoassay kit. The amount of cortisol was determined by measuring the absorbance at 450 nm as described above. All samples and standards were measured in duplicate, and the coefficient of variation was less than 5%. The salivary cortisol levels are expressed as µg/dl. Data was double-entered into an Epi Info 6.04d software application, and an internal consistency check was conducted. Statistical analyses were performed using the Stata Statistical Package, version 9. Descriptive statistics were used to report the socio-demographic information. Clinical characteristics of the sample used to assess infant neurobiological - motor development (i.e., the AIMS Score) were analyzed using one-way ANOVA. Bonferroni correction and correlations within the AIMS scores and biological markers were analyzed using a Spearman correlation. The Spearman correlation was used because the distributions of biomarkers did not fill the requirements for normality. The variables included in bivariate analyses were as follows: in the first level, social class, maternal age, type of delivery, infant sex, prematurity, birth weight, smoking, alcohol consumed in last year; in the second level, history of affective disorders, postpartum affective disorder (PPAD), type of PPAD episode, mother anxiety disorder, mother BDNF level, mother NGF level, mother IL-6 level, mother and infant cortisol levels. Linear regression of infant neurobiological - motor development (i.e., the AIMS Score) was used for all variables with a p-value≤0.2 when associated with exposure and outcome. We considered associations with a p-value≤0.05 to be statistically significant. Furthermore, to determine the grouping of the associated factors with infant motor development, we conducted an exploratory factorial analysis. The extraction method was Primary Component Analysis (PCA). The varimax rotation was used to facilitate the data interpretation, retaining independence of the factors. Only the variables that had statistical significance in the regression analysis were included. Factorial loadings greater than or equal to 0.3 were used to establish the factor to which each variable adhered. The Kaiser-Meyer-Olkin (KMO) test was conducted to verify sample adequacy in relation to the factorial analysis of primary components, which was adequate when greater than or equal to 0.5. In the same direction, Bartlett's test of sphericity was performed and was considered significant when less than 0.05. Finally, to obtain a fuller comprehension of the results, we performed another linear regression analysis with the generated factor scores in PCA. This work was approved by the Catholic University of Pelotas (UCPel) Ethics Committee 2008/30-2007/29 CONEP 44/2008-reg14253. The subjects provided written informed consent to their participation in this study. The infants were enrolled only if the mother or a legal guardian provided a written informed consent. The women with detected psychiatric disorders were referred to the Psychiatry Service at the UCPel. Infants with low motor development were referred to the UCPel physiotherapy clinic.
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