Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Dev Psychobiol . Author manuscript; available in PMC: 2026 Apr 14. Published in final edited form as: Dev Psychobiol. 2025 Nov;67(6):e70086. doi: 10.1002/dev.70086 Search in PMC Search in PubMed View in NLM Catalog Add to search Infant 6-month Psychophysiology During Interaction with Mother is Differentiated by 12-month Attachment Quality Bharathi J Zvara Bharathi J Zvara 1 Population & Biopsychosocial Health Innovations, School of Nursing, UNC-Chapel Hill, NC Find articles by Bharathi J Zvara 1 , Roger Mills-Koonce Roger Mills-Koonce 2 Department of Human Development and Family Science, School of Education, UNC-Chapel Hill, NC Find articles by Roger Mills-Koonce 2 , Cathi Propper Cathi Propper 3 Division of Maternal and Child Health, School of Nursing, University of North Carolina at Chapel Hill Find articles by Cathi Propper 3 , Karen Grewen Karen Grewen 4 Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC Find articles by Karen Grewen 4 , Brenda Pearson Brenda Pearson 5 Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC Find articles by Brenda Pearson 5 , Alison M Stuebe Alison M Stuebe 6 Division of Maternal-Fetal Medicine, UNC School of Medicine Find articles by Alison M Stuebe 6 Author information Copyright and License information 1 Population & Biopsychosocial Health Innovations, School of Nursing, UNC-Chapel Hill, NC 2 Department of Human Development and Family Science, School of Education, UNC-Chapel Hill, NC 3 Division of Maternal and Child Health, School of Nursing, University of North Carolina at Chapel Hill 4 Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC 5 Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC 6 Division of Maternal-Fetal Medicine, UNC School of Medicine ✉ Corresponding Author: Bharathi J. Zvara, University of North Carolina at Chapel Hill School of Nursing, Division of Maternal and Child Health, 4013 Carrington Hall, Chapel Hill, NC 27599-7460. [email protected] PMC Copyright notice PMCID: PMC13071831 NIHMSID: NIHMS2156095 PMID: 41015778 The publisher's version of this article is available at Dev Psychobiol Abstract Infant-mother attachment relationships play a crucial role in shaping children’s psychological and physiological well-being. This study examined whether attachment quality at 12 months is associated with infant psychophysiological responses to mild stress at 6 months. Participants were 222 ethnically and socioeconomically diverse mother-infant dyads followed from the third trimester of pregnancy through the infant’s first year. At 6 months, dyads participated in a free play session followed by the Face-to-Face Still-Face Paradigm (FFSFP). Infant saliva samples were collected before and after free play and at 1, 20, and 30 minutes post-FFSFP to measure oxytocin, cortisol, and salivary α-amylase, biomarkers associated with stress and social regulation. Maternal blood samples were collected at 10, 20, and 30 minutes post-FFSFP and analyzed for oxytocin and cortisol. Attachment quality was assessed at 12 months using the Ainsworth Strange Situation Paradigm. Linear mixed-effects models showed that securely attached infants had significantly higher oxytocin and lower sAA levels than insecurely attached infants, with sAA decreasing over time only in the secure group. No significant differences emerged in infant cortisol reactivity or maternal hormone levels. Findings suggest a potential link between infant attachment quality and stress regulation, particularly involving oxytocin and sympathetic nervous system activity. Keywords: Infant attachment, Psychophysiological response, Stress regulation, Oxytocin, Cortisol, α-Amylase Attachment behavior, considered an evolutionary adaptation, serves to modulate fear and distress through the proximal effects of physical and psychological contact with an attachment figure ( Bowlby, 1969 ). Such behaviors may occur not only during direct physical contact but also in the caregiver’s absence, as infants seek proximity or comfort through signaling, monitoring, or internal representations of the caregiver ( Bowlby, 1969 ). Individual differences in infant attachment are believed to represent distinct strategies for responding to interpersonal challenges (Ainsworth et al., 1978). Therefore, these differences are likely linked to the psychophysiological systems involved in managing interpersonal stress. Research increasingly focuses on the biological correlates of attachment, particularly as it relates to physiological responses to stress. The biological mechanisms of the stress response involve a complex interplay between several physiological systems, notably the autonomic nervous system (ANS), the hypothalamic-pituitary-adrenal (HPA) axis, and oxytocin. These systems collectively manage how the body responds to stressful experiences and social interactions, crucial for attachment and early development ( Numan & Young, 2016 ; Granger & Kivlighan, 2003 ). Physiological Systems and Stress Regulation The ANS, responsible for rapid physiological responses, prepares the body for immediate reactions such as “fight or flight” by increasing heart rate and blood flow. Its activation can be measured using salivary α-amylase (sAA), a marker that reflects the body’s response to both positive and negative stimuli ( Engert et al., 2011 ). In contrast, the HPA axis provides a slower but sustained response to stress by releasing cortisol, which helps regulate the ANS and maintain homeostasis. The coordination between these two systems is considered adaptive, promoting better health outcomes ( Tarullo et al., 2017 ; Hibel et al., 2020 ). Although both α-amylase and cortisol increase in response to challenge tasks, amylase peaks and recovers more quickly than salivary cortisol ( Nater et al., 2006 ). Additional research indicates that infants may show more pronounced increases in α-amylase compared to cortisol after mild stress tasks, such as the Still Face Paradigm (FFSFP, Tronick et al., 1978 ) or separation-reunion scenarios. In a sample of young children, Silke and colleagues (2025) identified distinct patterns of stress reactivity, showing that some children exhibited increasing sAA in response to mild stress, while cortisol responses were more variable and blunted. This aligns with prior research in adults ( Engert et al., 2011 , 2013) suggesting that sAA reflects the more immediate reactivity of the sympathetic nervous system (SNS), whereas cortisol, governed by the HPA axis, may respond more slowly and is more sensitive to chronic or intense stress exposure. Oxytocin, a neuropeptide produced in the hypothalamus, plays a significant role in social bonding and attachment, particularly during infancy ( Numan & Young, 2016 ). It is released during positive social interactions such as breastfeeding and affectionate contact with caregivers, fostering feelings of safety and security. Oxytocin also influences the stress response by dampening HPA axis activity, thereby reducing cortisol release and buffering the physiological impact of stress. However, while the role of oxytocin in stress response is well-documented in adults, its relationship with attachment security and stress regulation in infants remains less understood. Emerging evidence suggests that secure attachments are associated with higher oxytocin levels, potentially leading to more adaptive stress responses ( Gordis et al., 2006 ). The functioning of these systems, HPA axis (cortisol), SNS (sAA), and oxytocin, within the context of different attachment relationships remains an area of growing interest, particularly in understanding how they collectively influence stress responses in infants ( Granger & Kivlighan, 2003 ). Mother-Infant Attunement and Biobehavioral Synchrony Mother-infant attunement is fundamental to the development of infant attachment. Also referred to as synchrony, attunement is the reciprocal process through which caregivers and infants coordinate behavioral and physiological states, regulating each other’s emotions ( Bell, 2020 ; Abney et al., 2021 ). It is characterized by the mother’s ability to perceive, interpret, and respond appropriately to her infant’s cues, a process strongly associated with secure attachment ( Jonsson & Clinton, 2006 ). Theorists suggest that secure attachment fosters behavioral coordination, and biological synchrony between the caregiver and infant, forming a biobehavioral feedback system supporting the infant’s emerging self-regulation abilities ( Feldman, 2012 ; Hofer, 2006 ). From a psychophysiological perspective, biobehavioral synchrony is supported by foundational research showing early attachment relationships shape infants’ physiological regulation (e.g., Abney et al., 2021 ; Porges, 2001 ). Attunement, however, may vary based on attachment classification. Secure attachments are typically associated with higher levels of caregiver-infant synchrony, both behavioral and physiological, while insecure attachments are linked to disruptions in this regulatory process. For instance, Hill-Soderlund et al. (2008) reported that insecure-avoidant infants exhibited greater vagal withdrawal and elevated sAA during the Strange Situation. This suggests that despite outward calm, these infants experience heightened physiological stress, reflecting a breakdown in mother-infant attunement at both behavioral and biological levels. Research has demonstrated that infants’ physiological responses in the Strange Situation Paradigm (SSP) differ by attachment classification, with distinct patterns of stress reactivity and recovery observed across secure and insecure groups ( Hill-Soderlund et al., 2008 ; Smith et al., 2016 ; Groh & Narayan, 2019 ). For example, Barbosa et al. (2021) reported that distinct patterns of infant regulatory behavior observed during the FFSFP at 3 and 9 months were related to attachment classifications at 12 months. Fewer studies have explored whether early patterns of physiological regulation, observed in stressful caregiver-infant interactions like the Face-to-Face Still-Face Paradigm (FFSFP), predict later attachment organization. Empirical studies support the theoretical framework linking attachment quality to biobehavioral synchrony, showing that physiological markers of stress regulation vary as a function of attachment security. Securely attached infants tend to exhibit more adaptive hypothalamic-pituitary-adrenal (HPA) axis functioning, including lower basal cortisol levels and more efficient cortisol recovery following stress exposure ( Feldman, 2012 ). In contrast, insecurely attached infants show heightened or dysregulated cortisol responses, including elevated or prolonged reactivity ( Spangler & Grossmann, 1993 ). Similarly, salivary α-amylase (sAA), an indicator of sympathetic nervous system activation, is typically lower in securely attached infants during stress-inducing tasks compared to their insecure peers ( Hill-Soderlund et al., 2008 ). Collectively, this literature suggests that secure attachment supports more regulated physiological responses, while insecure patterns, particularly avoidant and disorganized, are linked to greater physiological dysregulation ( Gunnar & Donzella, 2002 ; Bernard & Dozier, 2010 ; Groh et al., 2012 ). The current study examines whether individual differences in infants’ physiological profiles (cortisol, sAA, and oxytocin) during the FFSFP at 6 months are associated with attachment classifications assessed at 12 months. The Current Study The majority of studies examining stress-related physiological responses in child development have focused on individual differences in HPA axis activity and cortisol reactivity to laboratory-induced stressors (for review, see Brindle et al., 2022 ). The current study investigates the association between physiological stress responses in infants at 6 months of age by measuring cortisol, oxytocin, and sAA levels during a well-validated controlled stress procedure, the FFSFP ( Tronick et al., 1978 ), and infant attachment quality measured at 12 months. We hypothesized that securely attached infants would show higher oxytocin, lower cortisol, and decreasing sAA levels, and that their mothers would exhibit lower cortisol and higher oxytocin in response to the FFSFP. Methods Participants We analyzed data from the Mood, Mother, and Infant Study (N=222). Mother-infant dyads were followed from the third trimester of pregnancy until 12 months postpartum. Women were recruited from prenatal and psychiatric clinics affiliated with a large university medical center in the Southeastern US and through email and social media between May 2013 and April 2017. Women with an elevated risk for postpartum depression and anxiety were oversampled, with risk ascertained through a Structured Clinical Interview for DSM-IV (SCID) at enrollment. Symptoms were coded as absent, subthreshold, or present based on DSM-IV criteria; in the full sample (N=222), 32% were rated as absent, 28.4% as subthreshold, and 39.6% as present. Eligible study participants were 18–45 years old, enrolled at >34 weeks’ gestation with a singleton pregnancy, able to communicate in English, intending to remain within 40 miles of the study site through the infant’s first birthday, and intending to breastfeed at least two months. Exclusion criteria included maternal diagnosis of Axis I disorders other than unipolar depression or anxiety disorders; current substance use; major congenital anomaly, NICU admission >12 h, or perinatal death; contraindication for breastfeeding; and current use of tricyclic antidepressants. The Mood, Mother, and Infant study was approved by the University of North Carolina Institutional Review Board (IRB 12–2016), and each participant provided written informed consent. Complete study details have been previously published ( Stuebe et al., 2019 ). Follow-up contacts occurred through monthly phone interviews and laboratory visits at 2, 6, and 12 months postpartum. Procedures Observational assessments During the 6-month postpartum lab visit, mother-infant dyads engaged in a 10-minute Free Play (FP) session, during which mothers were given a standard set of developmentally appropriate toys and instructed to interact with their infant as they normally would during unstructured play. Following this activity, dyads participated in the FFSFP, a widely used observational procedure designed to assess infant behavioral and physiological responses to social stress ( Mesman et al., 2009 ). Mothers were given a set of standardized instructions for each episode of the FFSFP (i.e., FF face-to-face, SF still-face, reunion). During the FFSF, the mothers were instructed to talk and interact with their child for 2 min normally (FF episode), then to turn away from the child for 15 s. After returning to face the child, the mothers were instructed to maintain a fixed stare, refraining from facial movements or display of affect for 2 min (SF episode). After turning head away for another 15 s, the mothers were instructed to again interact normally with the child for two-minutes. Negative child affect was demonstrated most often during the face-to-face episode and still-face episodes ( Ekas et al., 2013 ; Mesman et al., 2009 ). The FFSFP was stopped if the infant was unable to be soothed at any point during the procedure (or 15 seconds of heavy crying). The episodes were recorded to ensure the behaviors of mothers and infants could be observed and coded for the entire interaction. Infant affect was coded by a trained team of research assistants unaware of the hypotheses of the study. In separate viewings of the tapes, research assistants coded infant facial affect in 5-s intervals. Coders were trained to reliability using a large pool of pre-existing video recordings of FFSFP interactions. To assess interobserver agreement in the current study, 15% of the interactions were selected randomly and coded by a second coder. Reliability was calculated using kappa to correct for chance agreement (κ = 0.89). For biomarker assay purposes, infant saliva samples were collected before and after the Free Play (FP) session, as well as at 1-, 20-, and 30 minutes following completion of the FFSFP, and later assayed for oxytocin, cortisol, and sAA. Venous blood samples were drawn from mothers at 10-, 20-, and 30-minutes in the recovery period, for analysis of OT and CORT levels. At 12 months postpartum, mother-infant dyads completed the Ainsworth Strange Situation Paradigm (SSP; Ainsworth et al.,1978), a seven-episode observational procedure designed to assess infant attachment security. The SSP involves structured separations and reunions with the mother and a female stranger, allowing for the evaluation of the infant’s behavioral responses to stress and caregiver return. Each episode lasted 3 minutes, though separation episodes were shortened if the infant showed clear distress. SSP episodes were videotaped for subsequent coding. Two coders, including a criterion coder, were trained and certified in the SSP system by recognized experts at the University of Minnesota. Both coders were blinded to all participant information. Inter-observer reliability was measured using Cohen’s kappa (κ = .83). Any disagreements in the double-coded cases were resolved through discussion and consensus. Biomarker Assays Blood samples were collected into pre-chilled vacutainer tubes containing EDTA, and 500 KIU of aprotinin per milliliter of blood was added to inhibit proteinase activity and prevent peptide degradation. The samples were immediately centrifuged at 1,600 × g for 15 minutes at 4 °C to separate plasma. The resulting plasma was aliquoted into pre-chilled cryotubes and stored at −80 °C until assay. Infant salivary samples were collected using SalivaBio Infant Swabs (Salimetrics; State College, PA). After collection, the swabs were stored at −80 °C until analysis to preserve peptide integrity. Oxytocin levels in extracted plasma and saliva were measured using a commercial enzyme-immunoassay kit (Enzo Life Sciences), which relies on competition between endogenous oxytocin and oxytocin linked to alkaline phosphatase for antibody binding; after overnight incubation at 4 °C, a colorimetric reaction is developed and read after one hour, with assay sensitivity at 15.6 pg/mL and minimal cross-reactivity (<0.001) with similar neuropeptides. Plasma cortisol was quantified using a competitive radioimmunoassay (MP Biomedicals), where cortisol competes with radiolabeled hormone for antibody binding sites, with bound radioactivity measured via gamma counting; the assay has a sensitivity of 0.07 μg/dL and a standard range of 1–60 μg/dL. Salivary cortisol was assessed using a Salimetrics competitive enzyme immunoassay, where endogenous cortisol competes with horseradish peroxidase-conjugated cortisol for binding on a microtiter plate; detection involves a colorimetric reaction with tetramethylbenzidine and sulfuric acid, read at 450 nm, with a sensitivity below 0.007 μg/dL and a range of 0.007–1.8 μg/dL. sAA activity was measured using a kinetic enzyme immunoassay kit (Salimetrics), which quantifies α-amylase activity based on the enzymatic breakdown of a chromogenic maltotriose substrate, producing 2-chloro-p-nitrophenol detected at 405 nm over 3 minutes. Salivary cortisol was assessed using a competitive enzyme immunoassay kit (Salimetrics) following established protocols (Porter et al., 2003). Biomarker assays were performed in duplicate, and assay reliability was assessed by calculating intra- and inter-assay coefficients of variation (CVs). For infant salivary cortisol, the intra-assay CV was 3.11% and the inter-assay CV was 5.42%. For infant salivary alpha-amylase, the intra-assay CV was 7.45% and the inter-assay CV was 11.1%. For infant salivary oxytocin, the intra-assay CV was 9.94% and the inter-assay CV was 10.67%. Measures Coding infant attachment strategy. At 12 months, infant attachment was assessed via the Strange Situation Paradigm (SSP; Ainsworth et al., 1978), which involved coding behavioral responses to maternal separations and reunions. Digitally recorded videos were coded in accordance with the Ainsworth et al. classification system by coders who were blind to the participants’ characteristics (Ainsworth et al., 1978). Coders were certified in the SSP after training at the University of Minnesota, where they learned to classify attachment into secure, insecure-avoidant, insecure-resistant, or disorganized categories based on infant behavior. For analysis, infants were grouped into secure (B) versus insecure (A, C, D) categories. Covariates. To control for potential confounders, sociodemographic variables, including child sex and household income, were included as covariates in all regression models. We also controlled for maternaldepression using the Edinburgh Postnatal Depression Scale (EPDS; scores >13 indicate probable major depression), and anxiety was assessed using the State subscale of the State-Trait Anxiety Inventory (STAI). Results Overview of Analyses To examine physiological responses to a mild social stressor at 6 months and their relation to attachment classification at 12 months, we used linear mixed-effects models (LMMs) to assess changes in infant and maternal stress responses across the FFSFP. Models were estimated using the PROC MIXED procedure in SAS Version 9.4 (SAS Institute Inc., 2012). This analytic approach is well-suited for repeated measures designs (Little & Rubin, 1987) and handling missing data ( Schafer & Graham, 2002 ). Model parameters were estimated using restricted maximum likelihood (REML), and degrees of freedom were calculated using the between-within method. Sample characteristics can be found in Table 1 . Chi-square tests showed no significant differences between infants rated as secure or not secure regarding maternal age, education, child sex, ethnicity, or household income. At 12 months, attachment classifications were distributed as follows: 108 infants (57.1%) were classified as secure, 5 (2.6%) as avoidant, 21 (11.1%) as resistant, and 55 (29.1%) as disorganized. We examined differences in psychophysiological responses to a mild stress task across time points of the FFSFP at 6 months of age, and their association with attachment quality assessed at 12 months (classified as secure or insecure). Controlling for numerous covariates (ex., household income, maternal depression, and anxiety) we found that infants with secure attachment, on average, had higher oxytocin levels (adjusted means, secure infants =26.7, SE = 1.83; insecure infants =22.7, SE = 2.03, p = .11), and lower sAA (adjusted means, secure infants = 39.7, SE = 3.28; insecure infant =50.8, SE = 4.02, p = .03) across all assessment time periods compared to insecure infants. We also noted an effect of time, such that oxytocin levels increased ( p = 0.0001) and sAA decreased ( p = 0.0015) over assessment time points for both the secure and insecure infants ( Table 2 ). Table 1. Demographics of sample, Mean (Standard Deviation) or N (%), by attachment categories Sample Characteristics Secure N=108 Not Secure N=81 p-value * Maternal Age, years Mean (SD) 31.2 4.8 31.4 4.9 0.7256 Gestational Age, weeks Mean (SD) 39.7 1.1 39.6 1.2 0.3930 Baby’s sex Boy 57 52.8 41 50.6 0.7686 Girl 51 47.2 40 49.4 Ethnicity Non-Hispanic 97 89.8 74 91.4 0.7206 Hispanic 11 10.2 7 8.6 Race (5-categories) White 83 76.9 60 74.1 Black or African American 8 7.4 8 9.9 Asian 0 0.0 0 0.0 0.8951 Native Hawaiian/Pacific Islander 8 7.4 5 6.2 American Indian/Alaska native 9 8.3 8 9.9 Income (4-categories) < $24,999/YR 17 16.0 20 24.7 $24,000–39,999/YR 24 22.6 13 16.1 0.3426 $40,000–99,999/YR 20 18.9 18 22.2 > $100,000/YR 45 42.5 30 37.0 Parental Education (4-categories) <4 years college graduate 23 21.3 20 24.7 Graduated 4 years college 35 32.4 22 27.2 0.7080 Postgraduate 50 46.3 39 48.2 Marital Status Married 87 80.6 64 79.0 Living with partner 11 10.2 8 9.9 0.9157 Not married or living with partner 10 9.3 9 11.1 Employment Status Employed 79 73.2 62 76.5 0.5957 Not employed 29 269 19 23.5 Open in a new tab * p-values are from t-tests for continuous variables and chi-sq tests for categorical variables. Table 2. Least square means for biomarker levels of infants and mothers: results from linear mixed effects models adjusted for child sex, income, maternal depression, anxiety, and assessment timepoint. Secure group Insecure group P -value N Adjusted mean S.E. Adjusted mean S.E. Infants Oxytocin (pg/mL) 125 26.7 1.83 22.7 2.03 0.11 α-Amylase (U/mL) 102 39.7 3.28 50.8 4.02 0.03 Cortisol (μg/dL) 186 0.39 0.04 0.37 0.05 0.70 Mothers Oxytocin (pg/mL) 172 13.1 0.53 12.8 0.60 0.65 Cortisol (μg/dL) 177 6.0 0.23 6.1 0.26 0.72 Open in a new tab No significant differences emerged between secure and insecure infants with cortisol reactivity (p >.10); however, both groups decreased in cortisol over assessment time (p=.002). Mothers of securely and insecurely attached children did not differ in oxytocin (p = .62) or CORT (p = .75) levels, although both biomarkers decreased significantly over time (p < .0001 for both; see Figure 1 ). We conducted one additional model comparing infants with and without disorganized attachment. This analysis revealed no statistically significant differences across infant or maternal biomarkers. Figure 1. Least square means and 95% confidence intervals for biomarker levels of infants and mothers, based on linear mixed effects models adjusted for child sex, income, and maternal depression. Open in a new tab Infant timepoints: BL = Baseline; FP = Free play; R1 = 1 min post-FFSFP; R2 = 20 min post-FFSFP; R3 = 30 min post-FFSFP Maternal Oxytocin timepoints: FR = Free play; SF = Still Face; RU = Reunion; R1 = 20 min post-FFSFP; R3 = 30 min post-FFSFP Maternal Cortisol timepoints: BL1 = Baseline 1(before Free play); FP = Free play; BL2 = Baseline 2 (before FFSFP); FR = Free play; SF = Still Face; RU = Reunion; R1 = 20 min post-FFSFP; R3 = 30 min post-FFSFP. Discussion This study contributes to ongoing research on how biological systems influence individual differences in psychobiological stress regulation ( Hibel et al., 2020 ; Smith et al., 2016 ; Hill-Soderlund et al., 2008 ). Specifically, it explores biobehavioral functioning within the attachment system by examining three stress biomarkers, cortisol, sAA, and oxytocin, in secure and insecure infants and their mothers. Our findings suggest that early differences in infants’ self-regulatory processes, observed in dyadic interactions at six months, may be associated with attachment classifications at 12 months. These findings support and extend the work of Barbosa et al. (2021) , who found distinct regulatory behaviors during the FFSFP at 3 and 9 months were associated with attachment classifications at 12 months. The current study builds on this by providing physiological evidence through measures of cortisol, sAA, and oxytocin. Building on these findings, our study demonstrates that securely attached infants showed higher oxytocin levels across all assessment points at six months, consistent with more affiliative and prosocial physiological functioning ( Numan & Young, 2016 ; Granger & Kivlighan, 2003 ). In contrast, they exhibited lower levels of sAA, indicating reduced sympathetic nervous system activation and lower physiological arousal. These physiological patterns complement Barbosa et al.’s behavioral findings and align with prior research suggesting insecure attachment is associated with heightened stress reactivity by six months ( Hill-Soderlund et al., 2008 ). Although oxytocin and sAA levels differed by attachment classification, cortisol reactivity did not. This contrasts with prior studies linking insecure attachment to elevated cortisol responses ( Gunnar et al., 1996 ; Schieche & Spangler, 2005 ) and highlights the nuanced nature of early stress regulation. One possibility is that cortisol responds more gradually and may be more sensitive to prolonged or higher-intensity stress, rather than brief or mild challenges ( Tarullo et al., 2017 ; Hibel et al., 2020 ). In contrast, sAA, reflecting sympathetic nervous system activity, responds and recovers more rapidly, providing a more immediate index of physiological arousal ( Nater et al., 2006 ; Davis & Granger, 2009 ). Consistent with this, Silke et al. (2025) found sAA trajectories among young children varied systematically in response to stress and were more closely associated with maternal psychosocial risk factors than cortisol patterns, further highlighting sAA as a sensitive marker of individual differences in stress responsivity. Surprisingly, infant attachment was not significantly associated with maternal psychophysiological stress responses across assessment time points. While prior research supports the presence of physiological synchrony between mothers and infants, this process may be dynamic and develop over time. For instance, studies have documented mother-infant physiological attunement by six months, influencing emotional, hormonal, and autonomic regulation (Porter, 2022), though they have not specifically examined how this synchrony varies by attachment classification. These findings underscore the importance of early attachment relationships and infants’ stress regulation mechanisms, and they emphasize the need for further research to elucidate the underlying mechanisms driving these associations. Our post hoc analyses revealed no statistically significant differences in biomarker levels between infants with disorganized attachment and those without. This null finding may reflect the relatively small number of infants classified as disorganized, limiting statistical power. Additionally, disorganized behaviors reflect conflicting or unresolved stress responses, and their physiological patterns may vary widely across individuals in early infancy. Strengths of this study include its comprehensive longitudinal design, integration of observational paradigms (FFSFP and SSP), and use of multiple biomarkers (cortisol, oxytocin, sAA), offering a robust investigation of the relationship between attachment and stress physiology. Repeated biomarker sampling enabled the analysis of both immediate and recovery-phase stress responses, while mid-morning lab visits helped minimize diurnal variation. Several limitations must also be noted. First, collapsing across attachment subgroups may have obscured important differences, suggesting that future research should examine distinct attachment profiles separately. Additionally, the absence of significant differences in maternal psychophysiological stress responses based on infant attachment could indicate either a weaker link between maternal stress and infant attachment than hypothesized or limitations in our ability to detect subtle variations. The use of different biological specimens, saliva for infants and plasma for mothers, while appropriate for each group, may limit direct comparability of oxytocin and cortisol levels between them ( Koh & Koh, 2007 ). This study highlights the long-term link between infant attachment and physiological stress responses, emphasizing the need for comprehensive, culturally sensitive, and interdisciplinary research to fully capture the complex, reciprocal nature of attachment and its biological foundations. Acknowledgment(s) We would like to thank the families who participated in this study for their time and commitment. Data from this study came from the Mood, Mother and Infants study which was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01 HD073220-01). Footnotes Conflict of Interest The authors declare no conflicts of interest. Contributor Information Bharathi J. Zvara, Population & Biopsychosocial Health Innovations, School of Nursing, UNC-Chapel Hill, NC Roger Mills-Koonce, Department of Human Development and Family Science, School of Education, UNC-Chapel Hill, NC. Cathi Propper, Division of Maternal and Child Health, School of Nursing, University of North Carolina at Chapel Hill. Karen Grewen, Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC. Brenda Pearson, Department of Psychiatry, UNC School of Medicine, UNC-Chapel Hill, NC. Alison M. Stuebe, Division of Maternal-Fetal Medicine, UNC School of Medicine References Abney DH, DaSilva EB, & Bertenthal BI (2021). Associations between infant-mother physiological synchrony and 4- and 6-month-old infants’ emotion regulation. Developmental Psychobiology, 63(6), e22161. [ DOI ] [ PubMed ] [ Google Scholar ] Ainsworth MDS, Blehar MC, Waters E, & Wall SN (2015). Patterns of attachment: A psychological study of the strange situation. Psychology Press. [ Google Scholar ] Barbosa M, Beeghly M, Moreira J, Tronick E, & Fuertes M (2021). Emerging patterns of infant regulatory behavior in the Still-Face paradigm at 3 and 9 months predict mother-infant attachment at 12 months. Attachment & Human Development, 23(6), 814–830. 10.1080/14616734.2020.1757730 [ DOI ] [ PubMed ] [ Google Scholar ] Bauer AM, Quas JA, & Boyce WT (2002). Associations between physiological reactivity and children’s behavior: Advantages of a multisystem approach. Journal of Developmental and Behavioral Pediatrics, 23(2), 102–113. 10.1097/00004703-200204000-00007 [ DOI ] [ PubMed ] [ Google Scholar ] Bell MA (2020). Mother-child behavioral and physiological synchrony. Advances in Child Development and Behavior, 58, 163–188. [ DOI ] [ PubMed ] [ Google Scholar ] Bernard K, & Dozier M (2010). Examining infants’ cortisol responses to laboratory tasks among children varying in attachment disorganization: Stress reactivity or return to baseline? Developmental Psychology, 46(6), 1771. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Bowlby J (1969). Attachment and loss: Vol. 1. Attachment. Basic Books. [ Google Scholar ] Brindle RC, Pearson A, & Ginty AT (2022). Adverse childhood experiences (ACEs) relate to blunted cardiovascular and cortisol reactivity to acute laboratory stress: A systematic review and meta-analysis. Neuroscience & Biobehavioral Reviews, 134, 104530. [ DOI ] [ PubMed ] [ Google Scholar ] Davis EP, & Granger DA (2009). Developmental differences in infant salivary alpha-amylase and cortisol responses to stress. Psychoneuroendocrinology, 34(6), 795–804. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Dickerson SS, & Kemeny ME (2004). Acute stressors and cortisol responses: A theoretical integration and synthesis of laboratory research. Psychological Bulletin, 130(3), 355. [ DOI ] [ PubMed ] [ Google Scholar ] Ekas NV, Haltigan JD, & Messinger DS (2013). The dynamic still-face effect: Do infants decrease bidding over time when parents are not responsive? Developmental Psychology, 49(6), 1027–1035. 10.1037/a0029330 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Engert V, Vogel S, Efanov SI, Duchesne A, Corbo V, Ali N, & Pruessner JC (2011). Investigation into the cross-correlation of salivary cortisol and alpha-amylase responses to psychological stress. Psychoneuroendocrinology, 36(9), 1294–1302. 10.1016/j.psyneuen.2011.02.009 [ DOI ] [ PubMed ] [ Google Scholar ] Feldman R (2012). Parent-infant synchrony: A biobehavioral model of mutual influences in the formation of affiliative bonds. Monographs of the Society for Research in Child Development, 77(2), 42–51. [ Google Scholar ] Gordis EB, Granger DA, Susman EJ, & Trickett PK (2006). Asymmetry between salivary cortisol and alpha-amylase reactivity to stress: Relation to aggressive behavior in adolescents. Psychoneuroendocrinology, 31(8), 976–987. 10.1016/j.psyneuen.2006.05.010 [ DOI ] [ PubMed ] [ Google Scholar ] Granger DA, & Kivlighan KT (2003). Integrating biological, behavioral, and social levels of analysis in early child development research: Progress, problems, and prospects. Child Development, 74(4), 1058–1063. 10.1111/1467-8624.00589 [ DOI ] [ PubMed ] [ Google Scholar ] Grewen KM, Davenport RE, & Light KC (2010). An investigation of plasma and salivary oxytocin responses in breast- and formula-feeding mothers of infants. Psychophysiology, 47(4), 625–632. 10.1111/j.1469-8986.2009.00968.x [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Groh AM, & Narayan AJ (2019). Infant attachment insecurity and baseline physiological activity and physiological reactivity to interpersonal stress: A meta-analytic review. Child Development, 90(3), 679–693. 10.1111/cdev.13226 [ DOI ] [ PubMed ] [ Google Scholar ] Groh AM, Roisman GI, van IJzendoorn MH, Bakermans-Kranenburg MJ, & Fearon RP (2012). The significance of insecure and disorganized attachment for children’s internalizing symptoms: A meta-analytic study. Child Development, 83(2), 591–610. [ DOI ] [ PubMed ] [ Google Scholar ] Gunnar MR, Brodersen L, Nachmias M, Buss K, & Rigatuso J (1996). Stress reactivity and attachment security. Developmental Psychobiology, 29(3), 191–204. [ DOI ] [ PubMed ] [ Google Scholar ] Gunnar MR, & Donzella B (2002). Social regulation of the cortisol levels in early human development. Psychoneuroendocrinology, 27(1–2), 199–220. [ DOI ] [ PubMed ] [ Google Scholar ] Gunnar MR, Wewerka S, Frenn K, Long JD, & Griggs C (2009). Developmental changes in hypothalamus-pituitary-adrenal activity over the transition to adolescence: Normative changes and associations with puberty. Development and Psychopathology, 21(1), 69–85. 10.1017/S0954579409000054 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Hibel LC, Marceau K, & Buhler-Wassmann AC (2020). The transmission of psychobiological stress response systems across generations. Development and Psychopathology, 32(5), 1603–1624. 10.1017/S0954579420001185 [ DOI ] [ Google Scholar ] Hill-Soderlund AL, Mills-Koonce WR, Propper C, Calkins SD, Granger DA, Moore GA, & Cox MJ (2008). Parasympathetic and sympathetic responses to the strange situation in infants and mothers from avoidant and securely attached dyads. Developmental Psychobiology, 50(4), 361–376. [ DOI ] [ PubMed ] [ Google Scholar ] Hofer MA (2006). Psychobiological roots of early attachment. Current Directions in Psychological Science, 15(2), 84–88. [ Google Scholar ] Jonsson CO, & Clinton D (2006). What do mothers attune to during interactions with their infants? Infant and Child Development, 15(4), 387–402. [ Google Scholar ] Koh DSQ, & Koh GCH (2007). The use of salivary biomarkers in occupational and environmental medicine. Occupational and Environmental Medicine, 64(3), 202–210. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Mesman J, van IJzendoorn MH, & Bakermans-Kranenburg MJ (2009). The many faces of the Still-Face Paradigm: A review and meta-analysis. Developmental Review, 29(2), 120–162. 10.1016/j.dr.2009.02.001 [ DOI ] [ Google Scholar ] Nater UM, Rohleder N, Schlotz W, Ehlert U, & Kirschbaum C (2006). Determinants of the diurnal course of salivary alpha-amylase. Psychoneuroendocrinology, 31(5), 459–468. 10.1016/j.psyneuen.2005.10.011 [ DOI ] [ PubMed ] [ Google Scholar ] Numan M, & Young LJ (2016). Neural mechanisms of mother-infant bonding and pair bonding: Similarities, differences, and broader implications. Hormones and Behavior, 77, 98–112. 10.1016/j.yhbeh.2015.05.015 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Porges SW (2001). The polyvagal theory: Phylogenetic substrates of a social nervous system. International Journal of Psychophysiology, 42(2), 123–146. 10.1016/S0167-8760(01)00162-3 [ DOI ] [ PubMed ] [ Google Scholar ] Porter CL, Yang C, Jorgensen NA, & Evans-Stout C (2022). Development of mother-infant co-regulation: The role of infant vagal tone and temperament at 6, 9, and 12 months of age. Infant Behavior and Development, 67, 101708. 10.1016/j.infbeh.2022.101708 [ DOI ] [ PubMed ] [ Google Scholar ] Rohleder N, & Nater UM (2009). Determinants of salivary alpha-amylase in humans and methodological considerations. Psychoneuroendocrinology, 34(4), 469–485. 10.1016/j.psyneuen.2008.12.004 [ DOI ] [ PubMed ] [ Google Scholar ] Schafer JL, & Graham JW (2002). Missing data: Our view of the state of the art. Psychological Methods, 7(2), 147. 10.1037/1082-989X.7.2.147 [ DOI ] [ PubMed ] [ Google Scholar ] Schieche M, & Spangler G (2005). Individual differences in biobehavioral organization during problem-solving in toddlers: The influence of maternal behavior, infant-mother attachment, and behavioral inhibition on the attachment-exploration balance. Developmental Psychobiology, 46(4), 293–306. 10.1002/dev.20065 [ DOI ] [ PubMed ] [ Google Scholar ] Silke O, Simon SG, Sosnowski DW, Johnson SB, Granger DA, & Riis JL (2025). Patterns of stress-related change in salivary alpha-amylase and cortisol among young children: Associations with maternal psychosocial risk factors. Psychoneuroendocrinology, 171, 107221. doi: 10.1016/j.psyneuen.2024.107221 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Smith JD, Woodhouse SS, Clark CA, & Skowron EA (2016). Attachment status and mother-preschooler parasympathetic response to the Strange Situation Procedure. Biological Psychology, 114, 39–48. 10.1016/j.biopsycho.2015.12.009 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Spangler G, & Grossmann KE (1993). Biobehavioral organization in securely and insecurely attached infants. Child Development, 64(5), 1439–1450. 10.1111/j.1467-8624.1993.tb02963.x [ DOI ] [ PubMed ] [ Google Scholar ] Stuebe AM, Meltzer-Brody S, Propper C, Pearson B, Beiler P, Elam M, & Grewen K (2019). The Mood, Mother, and Infant Study: Associations between maternal mood in pregnancy and breastfeeding outcome. Breastfeeding Medicine, 14(8), 551–559. 10.1089/bfm.2019.0079 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Tarullo AR, John ER, & Gunnar MR (2017). Maturational changes in EEG power spectra in adolescence. Developmental Science, 20(5), e12423. 10.1111/desc.12423 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Tronick EZ, Als H, Adamson LB, Wise S, & Brazelton TB (1978). The infant’s response to entrapment between contradictory messages in face-to-face interaction. Journal of the American Academy of Child Psychiatry, 17, 1–13. 10.1016/S0002-7138(09)62273-1 [ DOI ] [ PubMed ] [ Google Scholar ] ACTIONS View on publisher site PDF (585.1 KB) Cite Collections Permalink PERMALINK Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Add to Collections Create a new collection Add to an existing collection Name your collection * Choose a collection Unable to load your collection due to an error Please try again Add Cancel Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top