# RISK AND PROTECTIVE FACTORS ASSOCIATED WITH EARLY ADVERSITY AND DEVELOPMENT: EVIDENCE FROM HUMAN AND ANIMAL RESEARCH

EDITED BY : Rosario Montirosso, Livio Provenzi and Ed Tronick PUBLISHED IN : Frontiers in Psychology and Frontiers in Behavioral Neuroscience

#### Frontiers eBook Copyright Statement

The copyright in the text of individual articles in this eBook is the property of their respective authors or their respective institutions or funders. The copyright in graphics and images within each article may be subject to copyright of other parties. In both cases this is subject to a license granted to Frontiers. The compilation of articles constituting this eBook is the property of Frontiers.

Each article within this eBook, and the eBook itself, are published under the most recent version of the Creative Commons CC-BY licence. The version current at the date of publication of this eBook is CC-BY 4.0. If the CC-BY licence is updated, the licence granted by Frontiers is automatically updated to the new version.

When exercising any right under the CC-BY licence, Frontiers must be attributed as the original publisher of the article or eBook, as applicable.

Authors have the responsibility of ensuring that any graphics or other materials which are the property of others may be included in the CC-BY licence, but this should be checked before relying on the CC-BY licence to reproduce those materials. Any copyright notices relating to those materials must be complied with.

Copyright and source acknowledgement notices may not be removed and must be displayed in any copy, derivative work or partial copy which includes the elements in question.

All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For further information please read Frontiers' Conditions for Website Use and Copyright Statement, and the applicable CC-BY licence.

ISSN 1664-8714 ISBN 978-2-88963-476-7 DOI 10.3389/978-2-88963-476-7

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

#### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# RISK AND PROTECTIVE FACTORS ASSOCIATED WITH EARLY ADVERSITY AND DEVELOPMENT: EVIDENCE FROM HUMAN AND ANIMAL RESEARCH

Topic Editors:

Rosario Montirosso, Scientific Institute IRCCS E. Medea, 0-3 Center for the at-Risk Infant, Bosisio Parini, Italy Livio Provenzi, Scientific Institute IRCCS E. Medea, 0-3 Center for the at-Risk Infant, Bosisio Parini, Italy Ed Tronick, University of Massachusetts Boston, & Harvard Medical School, United States

Citation: Montirosso, R., Provenzi, L., Tronick, E., eds. (2020). Risk and Protective Factors Associated With Early Adversity and Development: Evidence From Human and Animal Research. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-476-7

# Table of Contents

Chiara Sacchi, Pietro De Carli, Giovanni Mento, Teresa Farroni, Silvia Visentin and Alessandra Simonelli

*42 Adverse Behavioral Changes in Adult Mice Following Neonatal Repeated Exposure to Pain and Sucrose*

Manon Ranger, Sophie Tremblay, Cecil M. Y. Chau, Liisa Holsti, Ruth E. Grunau and Daniel Goldowitz

*56 Persistence of Effects of VLBW/PT Birth Status and Maternal Emotional Availability (EA) on Child EA Trajectories*

Dale M. Stack, Célia Matte-Gagné and Daniel J. Dickson

*71 Severe Perinatal Hypoxic-Ischemic Brain Injury Induces Long-Term Sensorimotor Deficits, Anxiety-Like Behaviors and Cognitive Impairment in a Sex-, Age- and Task-Selective Manner in C57BL/6 Mice but Can Be Modulated by Neonatal Handling*

Aida Muntsant, Kalpana Shrivastava, Mireia Recasens and Lydia Giménez-Llort

*90 Postweaning Isolation Rearing Alters the Adult Social, Sexual Preference and Mating Behaviors of Male CD-1 Mice*

Zi-Wei Liu, Yu Yu, Cong Lu, Ning Jiang, Xiao-Ping Wang, Shui-Yuan Xiao and Xin-Min Liu

*102 Effects of an Early Postnatal Music Intervention on Cognitive and Emotional Development in Preterm Children at 12 and 24 Months: Preliminary Findings*

Fleur Lejeune, Lara Lordier, Marie P. Pittet, Lucie Schoenhals, Didier Grandjean, Petra S. Hüppi, Manuela Filippa and Cristina Borradori Tolsa

*114 Prenatal Risk Factors for Adverse Developmental Outcome in Preterm Infants—Systematic Review*

Milla K. Ylijoki, Eeva Ekholm, Mikael Ekblad and Liisa Lehtonen

*126 Pain, Parental Involvement, and Oxytocin in the Neonatal Intensive Care Unit*

Manuela Filippa, Pierrick Poisbeau, Jérôme Mairesse, Maria Grazia Monaci, Olivier Baud, Petra Hüppi, Didier Grandjean and Pierre Kuhn

*141 Darwin's Other Dilemmas and the Theoretical Roots of Emotional Connection*

Robert J. Ludwig and Martha G. Welch

*165 Demographic and Parental Factors Associated With Developmental Outcomes in Children With Intellectual Disabilities*

Rosa Vilaseca, Magda Rivero, Rosa M. Bersabé, María-José Cantero, Esperanza Navarro-Pardo, Clara Valls-Vidal and Fina Ferrer

*180 Type of Delivery, Neuropsychological Development and Intelligence in Twin Births*

María-José González-Valenzuela, Ernesto González-Mesa, Olga Cazorla-Granados and Dolores López-Montiel

Anna-Lena Zietlow, Nora Nonnenmacher, Corinna Reck, Beate Ditzen and Mitho Müller

# Editorial: Risk and Protective Factors Associated With Early Adversity and Development: Evidence From Human and Animal Research

Livio Provenzi 1 \*, Rosario Montirosso1 and Ed Tronick 2,3

1 0-3 Center for the at-Risk Infant, Scientific Institute IRCCS E. Medea, Lecco, Italy, 2 Developmental and Brain Sciences Program, Department of Psychology, University of Massachusetts Boston, Boston, MA, United States, 3 Department of Newborn Medicine, Harvard Medical School, Boston, MA, United States

Keywords: adverse life events, behavioral epigenetics, brain, development, parenting, preterm birth, social touch, stress

**Editorial on the Research Topic**

#### **Risk and Protective Factors Associated With Early Adversity and Development: Evidence From Human and Animal Research**

#### Edited by:

Ilaria Grazzani, University of Milano Bicocca, Italy

#### Reviewed by:

Maria Spinelli, Università degli Studi G. d'Annunzio Chieti e Pescara, Italy

> \*Correspondence: Livio Provenzi

<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology

Received: 02 October 2019 Accepted: 09 December 2019 Published: 08 January 2020

#### Citation:

Provenzi L, Montirosso R and Tronick E (2020) Editorial: Risk and Protective Factors Associated With Early Adversity and Development: Evidence From Human and Animal Research. Front. Psychol. 10:2906. doi: 10.3389/fpsyg.2019.02906 The recent advances at the crossroad among behavioral science, psychoneuroendocrinology, neuroscience, genetics, and epigenetics are revealing how early environmental exposures—for good or ill—leave biochemical "scars" or signatures in our developmental biology. These biological and behavioral memorial processes of our precocious encounters with life constitute the interwoven mechanisms that contribute to the long-lasting programming of health and disease later in life. The quality of early caregiving (Weaver et al., 2004; Conradt et al., 2016; Lester et al., 2018) and the exposure to adverse life events (McCrory et al., 2010; Blaze et al., 2015; Provenzi et al., 2016)—even when the exposure occurs in previous generations (Yehuda et al., 2005; Moog et al., 2016)—are embedded into our developing phenotype and contribute to our health trajectories and disease risk from infancy to adulthood.

The rapid and consistent growth of this field is seen in the birth of new translationally relevant scientific areas, e.g., behavioral epigenetics (Lester et al., 2011), microbiome research (Robertson et al., 2019), and in new ramifications of existing and well-established areas, e.g., psychoneuroendocrinology (Heim et al., 2019), social and affective neuroscience (Cascio et al., 2019), behavioral neuroimmunology (Suárez-Álvarez et al., 2013). Future advances in these areas of scientific investigation will greatly contribute to our comprehension of the pathways of disease risk and inform smarter and more effective preventive and therapeutic early interventions, finally enabling us to use the environmental-sensitive nature of our genes and biology to serve neuroprotective goals (Heim et al., 2019).

From this perspective, in the present Research Topic, we aimed at collecting evidence from both animal models and human studies on these emerging research efforts which are directed at disentangling how these mechanisms (e.g., epigenetics, microbiome) may be "informed" by early environmental encounters and ultimately lead to specific health and disease outcomes. The 15 papers included in this issue vary widely in terms of subjects, exposures, mechanisms, and outcomes. The collection includes original research articles, theoretical viewpoints, systematic reviews, and methodological issues. Here, our hope was to make order out of this complexity by assuming a developmental view inspired by a dynamic system epistemological approach to early development (van Geert, 2008). The dynamic system approach is a theory of embodied and embedded actions that considers developmental processes as emerging from the continuous coupling between the organism and the environment. More specifically, the early, continuous and fine-tuned micro-regulatory processes occurring between the individual and the environment result in the establishment of stable, yet flexible attractor states that further contributes to define the developmental trajectories later in life (Smith and Thelen, 2003). In other words, development is considered as the byproduct of messy and reciprocal connections (and disconnections) between an individual and the physical and conspecific environment in which he/she is situated (Tronick, 2017). Environmental encounters—such as the quality of parental caregiving or the exposure to traumatic events—play a critical role as they contribute to the shaping of the complex weaving of connections that result in the emerging behavioral phenotype of a given individual. Notably, from this viewpoint, environmental encounters cannot be defined as positive or negative per sè. Rather, encounters may provide beneficial or detrimental contributions for the developmental trajectory of a living being depending on the biochemical, cognitive, and socio-emotional that embed their effects in the developing biology and behavior of that given individual. Most prominently, reparation processes at many levels of the individual—molecular to behavioral—of messy or disruptive encounters with the human and physical environment assume a critical role by increasing the complexity and coherence of these processes which ultimately contributes to better and healthier outcomes later in life (DiCorcia and Tronick, 2011).

The original articles published in this Research Topic highlight the potential of early adversities occurring from pregnancy (Zietlow et al.) to the perinatal period (Gonzalez-Valenzuela et al.; Muntsant et al.) and from childhood (Stack et al.; Vilaseca et al.) up to adulthood (Liu et al.; Ranger et al.). Moreover, evidence of the impact of positive caregiving on infants' brain development (Hanford et al.) and cognitive and behavioral outcomes (Lejeune et al.) has is also reported. Taken together, these findings highlight the messiness of the dynamic developmental framework in which a living being grows in complexity as well as the openness of the developing phenotype to a wide and diverse set of potential environmental encounters. These encounters range from perinatal brain damage (Muntsant et al.) to postnatal music exposure (Lejeune et al.), from parental sensitive caregiving (Stack et al.;Vilaseca et al.; Zietlow et al.) to painful stimulations (Ranger et al.). The exposure to such a variety of environmental exposures is capable of shaping of cognitive development (Lejeune et al.; Muntsant et al.), brain connectivity (Hanford et al.), emotional (Stack et al.), and neuroendocrine regulation (Zietlow et al.) and even social and mating behavior in adult life (Liu et al.).

The theoretical, review, and methodological papers collected in this Research Topic further contribute by putting emerging research in the field into a broader context and by providing a different perspective for interpreting the research findings and highlighting clinical implications. Ludwig and Welch discuss controversies in the Darwinian theoretical construction. They ambitiously propose two testable theoretical arguments that move from lessons learned in clinical practice with atrisk babies which inform a relational and epigenetic model of emotional development that can be beneficial for early assessment and interventions in infancy and childhood. Two reviews focus on the embedding of early life stress into the developmental trajectories of behavioral, neuroendocrine, and immune system regulation. The mini review from Fogelman and Canli underlines the neuroendocrine and immune system physiological processes which contribute to the long-term embedding of early environmental encounters. Ylijoki et al. systematically reviewed evidence of the prenatal risk factors involved in setting the risk for detrimental health outcomes in preterm infants. Filippa et al. further focus on a specific potential mechanism—oxytocin secretion and its regulation—that may help us in understanding how early parental engagement may be beneficial for healthy development of preterm infants exposed to repeated painful stimulation during the hospitalization in the neonatal intensive care unit. Sacchi et al. present a brilliant study protocol that holds potentials for revealing how the post-natal caring environment may be beneficial for the sociocognitive development in infants with intrauterine growth restriction, at least partially through neural mechanisms involved in social and non-social stimuli elaboration. Finally, a review of methodological approaches used in the field of developmental human behavioral epigenetics is provided by Provenzi et al., who highlight the pros and cons of different retrospective and prospective research architectures for the advancement of the field.

In conclusion, the present Research Topic successfully collected contributions from researchers and clinicians working in different European (i.e., Germany, Finland, Italy, Spain, Switzerland), Asiatic (i.e., China), and North American (i.e., Canada, United States of America) countries. The virtuous integration of expertise between the scientific and the clinical frameworks is hugely needed in order to advance the scientific knowledge in directions that can forcefully impact our capacity to provide smarter interventions and more efficient preventive strategies for at-risk individuals. We hope that the present Research Topic may be a step forward into this direction and that it can benefit future translational studies.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## FUNDING

This work was partially supported by grant RF-2016-02361884 to RM and RC2019 to LP from Italian Ministry of Health.

## REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Provenzi, Montirosso and Tronick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Impact of Caregiving on the Association Between Infant Emotional Behavior and Resting State Neural Network Functional Topology

Lindsay C. Hanford1 \*, Vincent J. Schmithorst2 , Ashok Panigrahy2 , Vincent Lee2 , Julia Ridley1 , Lisa Bonar1 , Amelia Versace1 , Alison E. Hipwell1 and Mary L. Phillips1

1 Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, PA, United States, 2 Department of Pediatric Radiology, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, United States

#### Edited by:

Livio Provenzi, Eugenio Medea (IRCCS), Italy

## Reviewed by:

Eleonora Mascheroni, Università Cattolica del Sacro Cuore, Italy Davide Crivelli, Università Cattolica del Sacro Cuore, Italy

> \*Correspondence: Lindsay C. Hanford<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology

Received: 24 May 2018 Accepted: 25 September 2018 Published: 15 October 2018

#### Citation:

Hanford LC, Schmithorst VJ, Panigrahy A, Lee V, Ridley J, Bonar L, Versace A, Hipwell AE and Phillips ML (2018) The Impact of Caregiving on the Association Between Infant Emotional Behavior and Resting State Neural Network Functional Topology. Front. Psychol. 9:1968. doi: 10.3389/fpsyg.2018.01968 The extent to which neural networks underlying emotional behavior in infancy serve as precursors of later behavioral and emotional problems is unclear. Even less is known about caregiving influences on these early brain-behavior relationships. To study brain-emotional behavior relationships in infants, we examined resting-state functional network metrics and infant emotional behavior in the context of early maternal caregiving. We assessed 46 3-month-old infants and their mothers from a community sample. Infants underwent functional MRI during sleep. Resting-state data were processed using graph theory techniques to examine specific nodal metrics as indicators of network functionality. Infant positive and negative emotional behaviors, and positive, negative and mental-state talk (MST) indices of maternal caregiving were coded independently from filmed interactions. Regression analyses tested associations among nodal metrics and infant emotionality, and the moderating effects of maternal behavior on these relationships. All results were FDR corrected at alpha = 0.05. While relationships between infant emotional behavior or maternal caregiving, and nodal metrics were weak, higher levels of maternal MST strengthened associations between infant positive emotionality and nodal metrics within prefrontal (p < 0.0001), and occipital (p < 0.0001) cortices more generally. Positive and negative aspects of maternal caregiving had little effect. Our findings suggest that maternal MST may play an important role in strengthening links between emotion regulation neural circuitry and early infant positive behavior. They also provide objective neural markers that could inform and monitor caregiving-based interventions designed to improve the health and well-being of vulnerable infants at-risk for behavioral and emotional problems.

Keywords: infant brain, resting state, neural network nodal metrics, emotion behavior, observed caregiving

## INTRODUCTION

fpsyg-09-01968 October 11, 2018 Time: 15:28 # 2

The rapid development of the human brain in the first years of life (Knickmeyer et al., 2008; Gao et al., 2015b) is important for brain-behavior relationships that set the stage for future clinical and functional outcomes. Sensorimotor, auditory and visual networks develop first (Lin et al., 2008; Liu et al., 2008), followed by other large-scale networks important for higherorder regulatory processes (Gao et al., 2009, 2015a), including: the default mode network (DMN), implicated in self-referential processing (Amsterdam, 1972; Gusnard et al., 2001; Raichle et al., 2001; Ramenghi et al., 2009; Short et al., 2013; Alcauter et al., 2014; Ball et al., 2015); the salience network, underlying attention to personally salient stimuli (Seeley et al., 2007; Akazawa et al., 2016); and the frontoparietal executive control network, important for multiple cognitive control processes including emotional regulation (Phillips et al., 2008; Gao et al., 2016). Additionally, the normal adult pattern of inverse correlation of resting state functional connectivity between the DMN and the dorsal attention network (Corbetta and Shulman, 2002; Akazawa et al., 2016) emerges in the first year (Gao et al., 2013).

Yet, links between early alterations in neural circuitry structure and function and clinically relevant behavioral outcomes remain unclear. In normally developing infants, there is heightened neural sensitivity to environmental factors, including caregiving behaviors (Sur and Rubenstein, 2005; Roth and Sweatt, 2011). These early experiences influence the formation of synapses and dendritic projections, which alter the recruitment of specific brain regions in largerscale networks, including those important for behavioral and emotional regulation (Huttenlocher, 1990; Kolb and Gibb, 2011). Although young infants have a rudimentary capacity to self-regulate, the expression and regulation of emotions is influenced by the provision of sensitive caregiving (Tronick and Gianino, 1986; Cirulli et al., 2003). Yet, the relationships between emotional network functioning and infant emotionality, and the impact of early caregiving on these relationships, are not well understood.

Positive and negative emotional behaviors (PE, NE) can be measured reliably within the first months of life (Worobey and Blajda, 1989; Rothbart et al., 2001; Dinehart et al., 2005). Infants displaying high levels of PE frequently smile or laugh, and experience high intensity pleasure (Tellegen, 1985; Watson et al., 1999). Some data suggest that low levels of PE in infancy precede behavioral inhibition (Park et al., 1997) and depression in childhood (Oldehinkel et al., 2004; Hayden et al., 2006; Anderson and Hope, 2008; Doucherty et al., 2010). Conversely, infants displaying high NE cry frequently and intensely in response to novelty and limitations, and are difficult to soothe (Henderson and Wachs, 2007). High NE in infancy is a relatively robust predictor of emotional problems later in childhood (Burgess et al., 2003).

Emotional regulation networks are well characterized in adults (Gray et al., 2002; Ochsner and Gross, 2005; Phillips et al., 2008), and include lateral and medial prefrontal cortical regions, specifically, anterior cingulate cortex (ACC), orbitofrontal cortex (OFC), and mediodorsal, ventrolateral, and dorsolateral prefrontal cortex (mdPFC, vlPFC, and dlPFC, respectively), within executive control and salience networks (Phillips et al., 2008; Sotres-Bayon et al., 2012; Nieh et al., 2013). Lower levels of functional connectivity between prefrontal cortical regions implicated in emotional regulation have been observed in children at risk for emotional disorders relative to healthy children (Singh et al., 2014; Manelis et al., 2015). By contrast, research examining relationships between largescale network function and emotional behaviors in infancy is minimal. One study reported greater OFC activity to sad versus neutral vocalizations in healthy 3- to 7-month-old infants (Blasi et al., 2011), suggesting an early functional specialization for processing human auditory NE. More recently, connectivity patterns of the amygdala in early infancy have been associated with developmental changes in temperament (Graham et al., 2016).

Parental caregiving plays a critical role in the development of infant emotional behaviors and emotional regulation (Tronick and Gianino, 1986; Oldehinkel et al., 2006; Thompson and Meyer, 2007). Caregiving behaviors can be subdivided into three distinct categories; positive, negative and mental-state talk (MST) (Park et al., 1997; Sharp and Fonagy, 2008; Lipscomb et al., 2011; Meins et al., 2012). Positive aspects of caregiving, including warmth and sensitivity, predict lower NE in offspring (Park et al., 1997), whereas more negative caregiving behaviors, such as harsh and intrusive behaviors, are associated with greater infant NE (Lipscomb et al., 2011). Longitudinal studies support the interaction between infant emotional reactivity and maternal caregiving in shaping later clinical and functional outcomes in offspring (Kochanska, 1995; Boyce and Ellis, 2005; Oldehinkel et al., 2006; Pluess and Belsky, 2010). Thus, high NE infants, who experience negative caregiving are at highest risk for later emotional dysregulation (Morrell and Murray, 2003), while high PE may act as a buffer in negative parenting environments (Lengua et al., 2000; Prior et al., 2001; Feder et al., 2009). Another aspect of caregiving, the mother's capacity to 'read,' understand and attribute mental states to her infant, is considered a prerequisite for sensitive caregiving (Sharp and Fonagy, 2008; Meins et al., 2012). Declarative indices of maternal MST have been linked with subsequent social–cognitive abilities in the child, including heightened emotion understanding (Doan and Wang, 2010). Our own work and that of others, has shown maternal MST to be a distinct aspect of maternal caregiving (Hipwell et al., 2016) with particular relevance for shaping emotional behaviors in the child (Meins et al., 2012; Hipwell et al., 2015).

Family environment influences on infant network function have been only briefly studied. Greater connectivity at rest between anterior and posterior regions of the DMN in 6– 12 months old infants was associated with higher levels of conflict between caregivers (Graham et al., 2015). This finding parallels connectivity patterns in depressed adults and children (Sheline et al., 2010; Gaffrey et al., 2012), suggesting a link between characteristics of the early caregiving environment, NE, and alterations in network connectivity in infancy. It further suggests negative caregiving may adversely affect network development in infancy and predispose to future mental health problems (Kochanska, 1995; Boyce and Ellis, 2005; Oldehinkel et al., 2006;

Pluess and Belsky, 2010). We are not aware, however, of any prior research that examined whether different types of caregiving behaviors impact neural networks, and relationships among networks and emotional behaviors, in infancy. Elucidating these relationships will be of clinical importance, as these relationships could help identify the most salient caregiving behavior targets in interventions designed to improve the health and well-being of vulnerable infants.

A robust method for establishing brain-behavior relationships is through network-based approaches, which can characterize the functional integration and segregation of large-scale networks (Supekar et al., 2009; Friston, 2011; Gao et al., 2015b). Two metrics commonly employed in such analyses are clustering coefficient (CC) and nodal efficiency (NEff). CC measures the degree to which nodes (neural regions) in a network segregate (Bullmore and Sporns, 2009; Rubinov and Sporns, 2010). Greater CC in network nodes is thought to provide more redundancy and render the network more resilient to insult to any single node (Bullmore and Sporns, 2009; Rubinov and Sporns, 2010). NEff measures the efficiency of communication of a single given node with other nodes in a network (Bullmore and Sporns, 2009; Rubinov and Sporns, 2010), and has been used to distinguish infants at-risk for future psychiatric problems including Autism Spectrum Disorder (Lewis et al., 2017). Using these networkbased approaches and nodal metrics during resting state allows examination of the specific nodal contributions to the segregation and/or efficiencies of large-scale network functional topology without requiring the use of cognitive tasks, and is thus wellsuited to studies in infants.

In the present study, we focused on infant network nodal metrics, infant displays of positive and negative emotional behaviors, and maternal caregiving behaviors at 3 months postpartum, as this represents a developmental window characterized by elemental forms of emotion regulation (Feldman et al., 1996; Horowitz-Kraus et al., 2017), and the appearance of early neural functional specialization for processing negative emotion (Blasi et al., 2011).

We aimed to:

nodal metrics in large-scale whole-brain networks. Based on findings linking positive caregiving and maternal MST with PE and self-regulation in the child (Meins et al., 2012), and findings linking higher NE and disrupted connectivity in emotional regulation networks in children at-risk for emotional problems (Singh et al., 2014; Manelis et al., 2015), we hypothesized that higher levels of positive caregiving and maternal MST would be associated with greater CC and NEff within emotion regulation networks. Specifically, we expected that these aspects of caregiving would strengthen positive relationships between infant PE, CC and NEff, and weaken inverse relationships between infant NE, CC and NEff, within these networks. By contrast, more negative caregiving would be associated with lower CC and NEff, and would strengthen inverse relationships between NE, CC and NEff in these networks.

## MATERIALS AND METHODS

#### Participants

Mothers (aged 19–24 years) and their 3-month-old infants were recruited from the population-based Pittsburgh Girls Study (PGS), an ongoing longitudinal study which has conducted annual assessments on 2,450 girls followed from childhood through young adulthood (Hipwell et al., 2002; Keenan et al., 2010). Pregnant or recently delivered participants were identified from PGS interview data in waves 15–17, and study eligibility was confirmed by telephone. Following written, informed consent for their own and their infant's participation in the study, motherinfant dyads completed a research visit at the Children's Hospital of Pittsburgh. Procedures were approved by the University of Pittsburgh Institutional Review Board.

Exclusion criteria for the mother included prenatal or concurrent substance exposure (except for marijuana use), and provision of <2 h per day of care for her infant. Exclusion criteria for infants included: <37 weeks gestation, head circumference <32 cm, birth weight <5.5 lbs, APGAR score <7 at 5 min, extended hospitalization for any reason, or any MRI contradictions.

## Demographic and Clinical Information

Mothers reported on infant age and gender. Mothers also reported on their own mood in the past week using the Edinburgh Postnatal Depression Scale (EPDS) (Cox et al., 1987), given the potential impact of maternal depression on caregiving (Wisner et al., 2002; Logsdon et al., 2006). Household poverty was indicated by maternal report of ten potential sources of public assistance (e.g., food stamps, Medicaid, and WIC). Low levels of poverty were indicated by receipt of 0–2 types of assistance, whereas moderate to high levels were indicated by three or more types.

#### Behavioral Assessments

Each mother-infant dyad was filmed in face-to-face interaction. The mother was first asked to talk to her infant "in any way you want to" without the use of toys (2 min), and then to "help

your child to get interested" in a specific toy (3 min). Infant and caregiving behaviors during the two episodes were coded independently, using time-sampled global ratings, by pairs of trained coders at graduate and doctoral level who were unaware of all other information about the participants.

Positive and negative infant emotional behaviors were coded on 5-point behaviorally anchored Likert scales ranging from 1 (none) to 5 (frequent, intense, prolonged displays). Mean scores across the two observation periods were computed for infant PE and NE.

Maternal behaviors were coded on five global dimensions (hostility, intrusiveness, involvement, warmth and sensitivity) scored on 4-point Likert scales ranging from 1 (none/minimal) to 4 (many/high) adapted from the Early Parenting Coding System (Shaw et al., 2006; Hipwell et al., 2015). Mean scores on each dimension were computed from observations in the two episodes. A varimax rotation principal component analysis of these mean caregiving scores resulted in two components: positive maternal caregiving (warmth, involvement, sensitivity), and negative maternal caregiving (hostility, intrusiveness) that explained 74.9% of the variance.

Frequency of maternal MST was determined from verbatim transcriptions of maternal speech during the filmed interactions. Comments about infant internal states or intentionality (e.g., Do you think that's funny? and Do you want to go home?) were counted and reduced to 4-point scales (1 = none to 4 = five or more comments), and a mean maternal MST score from the two observation episodes was computed.

Inter-rater reliability for coded observations was examined in a random selection of 15 (26%) dyads. Intraclass correlation coefficients (ICCs) computed for absolute agreement on coded behavioral observations were high: 0.88 infant PE; 0.85 infant NE; 0.76 maternal hostility; 0.88 maternal intrusiveness; 0.91 maternal involvement; 0.85 maternal warmth; 0.70 maternal sensitivity; and 0.89 for maternal MST.

### Resting State Data Acquisition and Image Processing

Infants were scanned during natural sleep without sedation (Haney et al., 2010; Windram et al., 2012). Mothers were asked to refrain from feeding or allowing their infant to nap for a few hours before the scan appointment, then at the allotted time the infant was swaddled and fed ('feed and bundle' approach) in a quiet, dimly lit room and then transferred to the MR scan table when asleep. Mothers without MRI contraindications could stay with the infant in the scan room.

Two 5-min resting state functional magnetic resonance images were acquired using a gradient-echo echo-planar imaging scan sequence with the following parameters: repetition time = 2020 ms, echo time = 32 ms, field of view = 256, 32 slices, and voxel size = 4 × 4 × 4 × mm3 .

Images were preprocessed using in-house routines, the Cincinnati Children's Hospital Imaging Processing Software (CCHIPS) (Schmithorst et al., 2000) written in IDL (Research Systems Inc., Boulder, CO), Statistical Parametric Mapping (SPM8) software1 , and the Brain Connectivity Toolbox (BCT) software (Rubinov and Sporns, 2010). Image preprocessing included slice time correction, motion correction, and a 2-step normalization procedure. Motion correction methods closely followed those laid out by Power and colleagues in order to avoid spurious results due to excessive motion (Power et al., 2014). Motion correction was performed using an affine transform to the best reference image within each resting state run. The reference image was selected by determining the frame within each run that best minimized an intensity-based cost function (Power et al., 2014). A two-stage procedure for spatial normalization was used: one, the reference image for each participant was normalized to an age-specific T2-weighted image (Shi et al., 2011) in MNI space, and two, all normalized reference images were then averaged to create a study-specific template, and then used as a normalization template for each participant's specific reference image. Finally, the entire run was transformed into MNI space (using a single transformation incorporating the motion correction parameters) and intensity normalized to grand mean = 1000. Frames with an rms deviation (DVARS) of greater than 25 from the previous frame, or a total movement from a framewise displacement (FD), as estimated from the motion correction parameters, of greater than 0.2 mm from the previous frame were rejected.

Data from each of the two resting state runs were combined into a single time course. Given the rigor of the above thresholds, a threshold of at least 4 min of total data (incorporating both runs), or 3.6 min of total data (with just one run) was used to include as many participants as possible. The number of usable frames was calculated and included as a covariate of no interest. An age-specific parcellation atlas (Shi et al., 2011) was used to extract average time courses from 90 bilateral cortical regions. Each time course was band-pass filtered (0.009 Hz < f < 0.08 Hz), and nuisance regressors including motion correction parameters, linear and quadratic drift, and global, average white matter and CSF signal, were used. Correlation matrices (90-X-90, absolute value) were computed based on the correlations of the time courses between two regions after removal of nuisance regressors and band-pass filtering.

The correlation matrices were thresholded (yielding a binarized graph, where each possible connection between nodes has a value of either zero or one) and computed to obtain specific nodal measures. The matrices were thresholded according to differing values of cost (the ratio of the number of connections in the graph to the number of possible connections). A costindependent analysis was used according to fixed values of cost ranging from 0.05 to 0.45 (step size 0.05). Here, subject was included as a random variable within these random slopes models, since thresholding at different cost values would be expected to have a multiplicative and not just a linear effect on the graph metrics. These models were averaged to obtain a more stable value per subject.

The formulae for CC and NEff are given below, for the binarized graphs used here. The connection between nodes i and j in the graph is designated as Gij, which has a value of zero or

1http://www.fil.ion.ucl.ac.uk/spm

one. The shortest distance (or path length) between nodes i and j is designated as Dij; if the nodes are completely unconnected, the distance is infinite. The clustering coefficient of a node i is defined as:

$$CC\_i = \frac{\sum\_{j,k} G\_{ij} G\_{ik} G\_{jk}}{\sum\_{\substack{j,k\\j \neq k}} G\_{ij} G\_{ik}}$$

and can be interpreted as the ratio of "triangles" (e.g., nodes i, j, and k are all connected to each other) to the number of possible "triangles" (e.g., node i is connected to nodes j and k). The nodal efficiency of a node i is defined as:

$$N \text{e} \mathcal{F}\_i = \frac{1}{N - 1} \sum\_{j} \frac{1}{D\_{ij}}$$

where N is the total number of nodes in the graph, and 1/Dij = 0 for the case where Dij is infinite (the nodes are unconnected). This metric is the average "efficiency" (reciprocal path length) of node i for communicating with all the other nodes in the graph.

#### Statistical Analyses

See **Figure 1** for study design and statistical analysis structure.

**Aim 1:** Associations among specific nodal metrics (CC, NEff) and infant behaviors were examined in two separate regression models: where whole-brain nodal metrics were dependent variables, and either NE or PE, were independent variables.

**Aim 2:** Associations among specific nodal metrics (CC, NEff) and positive caregiving, negative caregiving or MST were examined in three regression models: where whole-brain nodal metrics were dependent variables, and either positive caregiving, negative caregiving or MST were independent variables. Next, the moderating effect of maternal caregiving on associations between infant NE and PE and specific nodal metrics was explored in six regression analyses that each included an interaction term: NE x negative caregiving, NE x positive caregiving, NE x maternal MST, PE x negative caregiving, PE x positive caregiving or PE x maternal MST, covarying for main effects of the corresponding infant and maternal caregiving measures.

In each set of analyses, infant age, gender, maternal depressed mood, use of public assistance, and number of usable frames, were covariates of no interest (**Supplementary Materials** for main effects of these covariates). Results were FDR corrected at alpha = 0.05. A mixed-effects model with an unstructured correlation matrix was used to account for the varying values of cost.

All interactions that survived multiple comparisons correction were graphed using ggplot2 (Wickham, 2016) in R2 to better display the relationships between infant emotional behavior, nodal metrics and maternal MST.

2https://www.r-project.org/

FIGURE 1 | Study design and data analyses. Three-month infants were scanned during natural sleep without sedation. Mother-infant dyads were filmed in a face-to-face interaction in order to code global ratings of infant emotional behavior and maternal caregiving. Data processing involved a standard pipeline: motion correction, normalization and smoothing. Images were segmented into 90 whole brain regions of interest (ROI) based on the aal neonatal atlas. Graph theory techniques defined the interactions between nodes, specifically, clustering coefficient and nodal efficiency. Analyses determined relationships among nodal network metrics and measures of infant emotionality, and the moderating effects of maternal caregiving on these relationships. Informed written consent was obtained for the publication of mother and infant images.

## RESULTS

Resting state fMRI and observational measures of infant emotional behaviors and maternal caregiving were collected for 58 infants. Ten infants were excluded from analyses due

#### TABLE 1 | Demographic, clinical, behavioral, and other covariate information.

∗Social assistance tally was measured by counting the number of public assistance services used, these were further dichotomized into low (0–2 counts) or high (3–7 counts).

‡An EPDS score >10 was reported by 20% of mothers, indicating 'probable' depression, while an EPDS score of >13 was reported in 12.5%.

to excessive motion, and two mothers did not complete the EPDS. Thus, 46 infants were included in total (**Table 1**). For 67% of mothers, this was their first born infant, for 27% this was their second child. For the remaining 6%, the birth order ranged from third to fifth child. The majority of mothers (76%) reported using 3–7 counts of public assistance. 20% of mothers indicated 'probable' postnatal depression on the EPDS (a score of >10 on the EPDS), and 12.5% had EPDS scores indicative of postnatal depression (score of >13), consistent with population norms (O'hara and Swain, 1996; Wisner et al., 2002).

Infant emotional and maternal caregiving variables are in **Table 1**. Infant negative and positive emotional behaviors were uncorrelated (rs = −0.07, ns), as were positive and negative caregiving behaviors with MST (rs = 0.29, ns and rs = −0.14, ns, respectively).

### Network-Emotional Behavior and Network-Caregiving Relationships

No relationships between either PE or NE and CC or NEff survived FDR correction, nor did relationships between maternal positive caregiving, negative caregiving or maternal MST and CC or NEff (**Table 2**).

TABLE 2 | Network nodal metric associations with positive and negative infant emotional behaviors and with maternal caregiving indices.

CC, clustering coefficient; NEff, nodal efficiency.

No metrics survived FDR <0.05 correction for multiple comparisons.

#### Impact of Caregiving on Network-Emotional Behavior Relationships

There was one significant positive moderating effect of positive maternal caregiving on the relationship between PE and NEff in the left middle OFC. This PE-NEff relationship was less inverse with higher levels of positive maternal caregiving (**Table 3**). There was one significant positive moderating effect of negative maternal caregiving on the relationship between infant NE and CC in the right inferior parietal lobule. This NE-CC relationship was more positive with higher levels of negative maternal caregiving (**Table 3**).

Several highly significant FDR-corrected moderating effects of maternal MST were observed for the relationship between PE and CC. Here, there were robust positive moderating effects of MST on PE-CC relationships in bilateral OFC, left middle and inferior frontal gyri, bilateral olfactory cortex of the OFC, bilateral medial superior prefrontal cortex, left rectus gyrus, bilateral insula, left ACC, bilateral calcarine cortex, bilateral cuneus, left lingual, bilateral superior and middle occipital gyri, right inferior parietal lobule, bilateral precuneus, and left superior and right middle temporal pole. In each case, stronger positive PE-CC relationships were shown at higher levels of maternal MST, while these relationships were weakened, becoming less positive or inverse, at lower levels of MST (**Table 4** and **Figure 2**). Negative moderating effects of maternal MST were observed for the relationship between PE and CC in the right supramarginal gyrus and between PE and NEff in the left superior temporal gyrus. Here, inverse PE-CC relationships were weakened or became positive with higher maternal MST (**Table 4**).

The moderating effects of maternal MST did not survive FDR correction for relationships between infant NE and either CC or NEff (**Table 4**).

TABLE 3 | Moderating effects of positive and negative maternal caregiving on the associations between nodal metrics and positive and negative infant emotional behaviors.

CC, clustering coefficient; NEff, nodal efficiency.

∗Denotes results that survived FDR <0.05 correction for multiple comparisons. A positive interaction denotes that the relationship between nodal metrics and infant emotionality was more positive (or less negative) with greater level of the given maternal caregiving behavior. A negative interaction denotes that the relationship between nodal metrics and infant emotionality was more negative (or less positive) with greater levels of the given maternal caregiving behavior.

#### DISCUSSION

We aimed to identify large-scale neural network-emotion relationships in 3-month-old infants, and examine the impact of caregiving on these relationships. Our major findings indicate that maternal MST influenced associations between infant PE and CC predominantly in prefrontal and occipital cortical networks, where higher levels of maternal; MST were associated with stronger positive relationships between infant PE and CC within these networks. These results contrast with more limited effects of positive and negative components of caregiving on infant brainemotional behavior relationships; and only weak relationships among nodal metrics and either maternal caregiving or infant emotional behaviors when examined without consideration of the caregiving context. These findings suggest a specific and critical role of maternal MST in shaping positive relationships between the extent of segregation, and resulting resilience, within prefrontal cortical and visual processing networks and PE in infants.

Higher levels of maternal MST were associated with stronger positive relationships, and weaker inverse relationships, between infant PE and CC among several lateral and medial prefrontal cortical regions implicated in emotional regulation (Gray et al., 2002; Ochsner and Gross, 2005). The lateral prefrontal cortex integrates emotional and cognitive information, and generates emotional responses (Gray et al., 2002). The medial prefrontal cortex, including the OFC, serves a critical role in memory retrieval, action selection, executive control and TABLE 4 | Moderating effects of maternal mental-state talk on the associations between nodal metrics and positive and negative infant emotionality.

CC, clustering coefficient; NEff, nodal efficiency; ∗denotes results that survived FDR <0.05 correction for multiple comparisons. §Graphical representation of these metrics available in Figure 1.

A positive interaction denotes that the relationship between nodal metrics and infant emotionality was more positive (or less negative) with greater maternal mentalstate talk. A negative interaction denotes relationships between nodal metrics and infant emotionality that were more negative (or less positive) with greater maternal mental-state talk.

social cognition related to emotional regulation (Ochsner and Gross, 2005). Greater maternal MST was also associated with stronger positive relationships between PE and CC in bilateral insula, important for interoception and a key role in the salience network (Zaki et al., 2012; Simmons et al., 2013), and in visual cortical regions important for

were less positive or inverse at lower levels of mental-state talk.

higher-order visual processing. Mothers' capacity to engage in mentalizing behaviors is linked to behavioral and neural indices of empathy and reflection, and may also be a proxy for mother-infant dyadic attunement (Meins et al., 2012). We show the importance of observed maternal MST in facilitating infant positive emotion and higher levels of segregation and built-in redundancy and resilience in emotional regulation, interoception, salience and visual processing networks in early infancy.

There were negative moderating effects of maternal MST on CC and NEff in the right supramarginal gyrus and the left superior temporal cortex, respectively, where higher levels of maternal MST were associated with stronger inverse relationships between infant PE and CC and NEff. The right supramarginal gyrus is important for somatosensory processing and empathy (Reed and Caselli, 1994; Carlson, 2012; Silani et al., 2013), while the left superior temporal gyrus is critical for language, speech, and higher-level auditory processing (Bigler et al., 2007). Given the impact of maternal MST on infant brain-PE relationships described above, maternal MST may obviate the need for either the early segregation of neural networks implicated in somatosensory and empathic processing, or the integration of neural networks subserving higher-level auditory processing. Instead, this characteristic of caregiving may promote involvement of large-scale prefrontal executive control and visual networks in the shaping of infant brain-PE relationships.

There were two significant relationships relating to moderating effects of positive and negative caregiving. More positive maternal caregiving weakened the inverse relationship between PE and NEff in the left OFC, indicating that greater, rather than lower, engagement of this key emotional regulation prefrontal cortical region may help to promote positive emotion in infants in supportive caregiving environments. More negative

maternal caregiving rendered the relationship between NE and CC in the right inferior parietal lobule positive. The latter region is important for directing attention to salient new or alerting environmental stimuli (Lynch et al., 1977; Singh-Curry and Husain, 2009), and thus a positive relationship between NE and CC in this region likely reflects enhanced attention to potentially alerting or threatening stimuli in infants with higher levels of NE. That this relationship was strengthened in more negative caregiving environments parallels reports showing an effect of negative caregiving in promoting greater vigilance and NE in infants (Lipscomb et al., 2011).

The absence of strong relationships among maternal caregiving behaviors and nodal metrics per se, or among infant emotional behaviors and nodal metrics outside the caregiving environment, indicate a specific effect of maternal caregiving in shaping early infant brain-emotional behavior relationships. Our findings also parallel those of other studies indicating effects of positive and sensitive caregiving in providing normative social, cognitive and emotional contexts for the development of self-regulatory capabilities, and greater higherorder functioning within these domains (Bick and Nelson, 2016). Studies examining the absence of positive caregiving, including maternal separation studies, and subsequent interventions, help to elucidate caregiving effects on neural networks subserving emotional regulation (Brett et al., 2015; Bick and Nelson, 2016). Here, studies of institutional care and adoption highlight the importance of caregiving on development of child neural networks (Gee et al., 2013; Bick and Nelson, 2016). The critical role of maternal caregiving in shaping infant behavior is further highlighted in rodent studies that provide links between levels of maternal attention, through extensive licking and grooming behaviors and typical rat pup development, and the development of anxiety-like behaviors in rat pups who are ignored by their mothers (Meaney, 2001). Moreover, cross-fostering studies show these latter behavioral changes can be reversed by reintroducing pups to highly attentive foster mothers (Francis et al., 1999; Weaver et al., 2004). Non-human primate studies demonstrate that social behaviors and amygdala gene expression are directly impacted by quality of maternal care (Sabatini et al., 2007). While these studies highlight the sensitivity of the early developmental period in humans, non-human primates and rodents to effects of caregiving on emotional behaviors and neural network development, our findings are the first to show a specific impact of caregiving on shaping critical neural network-emotion relationships in human infants.

The study has some limitations. The cross-sectional nature of analyses does not provide information on the direction of relationships between maternal caregiving, infant emotion and nodal metrics. Given that this is the first study to explore any relationships among these variables, however, identifying associations is a logical first step. The relatively small sample size limited our ability to examine the influence of additional environmental factors (e.g., maternal psychiatric history) on relationships among neural measures and emotional behavior. Similarly, our sample was predominantly of low SES, which limited our ability to generalize to other populations. Low SES samples are often under-represented in research, however, and thus inclusion of such samples in future infant studies will allow examination of a greater range of emotional developmental outcomes. We did not include birth order as a covariate. This can be an additional focus of study in future research. Longitudinal studies can establish the temporal ordering of associations among infant behavior, brain functional topology and caregiving within a larger sample.

We are the first to show that maternal MST, a key component of parenting linked to emotional behavior in offspring, influences the association between infant positive emotion and functional topology in prefrontal cortical and visual processing networks. Moreover, these associations were weak in the absence of moderating effects of any caregiving components. These findings thus highlight the importance of MST, above positive or negative aspects of caregiving, on development of infant brain-positive emotional behavioral relationships. These findings are clinically important, as they can provide objective neural markers to monitor the effectiveness of caregiving-based interventions targeted at strengthening infant brain-positive emotion relationships, to improve the health and well-being of vulnerable infants at risk for behavioral and emotional problems.

## ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the University of Pittsburgh Institutional Review Board. The protocol was approved by the University of Pittsburgh Institutional Review Board. All mothers gave written informed consent their own and their infant's participation in the study in accordance with the Declaration of Helsinki.

## AUTHOR CONTRIBUTIONS

MP and AH conceived and designed experiments. JR, AH, and LH performed all the experiments. LH, VS, AH, and MP analyzed the data. LH, AH, and MP wrote the manuscript with input from all authors.

## FUNDING

This work was supported by NIMH R21 MH106570 (Phillips, Hipwell) and The Pittsburgh Foundation (Phillips).

## ACKNOWLEDGMENTS

We thank the participants and families for their efforts to take part in this study.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg.2018. 01968/full#supplementary-material

## REFERENCES

fpsyg-09-01968 October 11, 2018 Time: 15:28 # 10

Carlson, N. R. (2012). Physiology of Behavior, 11th Edn. London: Pearson.

and antisocial behaviour. Crim. Behav. Ment. Health 12, 99–118. doi: 10.1002/ cbm.489

study from 2 months to 8 years. J. Child Psychol. Psychiatry 44, 489–508. doi: 10.1111/1469-7610.t01-1-00139

Disorders, ed. A. H. T. J. D. Maser (Hillsdale, NJ: Lawrence Erlbaum Associates, Inc), 681–706.

**Conflict of Interest Statement:** The sponsor had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Copyright © 2018 Hanford, Schmithorst, Panigrahy, Lee, Ridley, Bonar, Versace, Hipwell and Phillips. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Methodological Challenges in Developmental Human Behavioral Epigenetics: Insights Into Study Design

Livio Provenzi 1 \*, Maddalena Brambilla1 , Renato Borgatti 2 and Rosario Montirosso1

1Scientific Institute, IRCCS Eugenio Medea, 0-3 Center for the at-Risk Infant, Bosisio Parini, Italy, 2Scientific Institute, IRCCS Eugenio Medea, Neuropsychiatry and Neurorehabilitation Unit, Bosisio Parini, Italy

Developmental human behavioral epigenetics (DHBE) holds potential for contributing to better understanding of how early life exposures contribute to human developmental trajectories and to inform clinical practice and early interventions. Nonetheless, DHBE research to date is challenged by two major issues: (a) the frequent use of retrospective study designs; and (b) the major focus on epigenetic variations associated with early life adversities, rather than protective care exposures. In order for DHBE research to maintain its promises, these issues need to be addressed in a systematic way according to a careful methodological planning of study design. In this contribution, we provide pragmatic insights on methodological aspects that should be dealt with while designing DHBE studies. We propose different study designs for the retrospective and prospective investigation of both adversity- and care-related epigenetic variations. Examples from available scientific literature are provided to better describe the advantages and the limitations of each study design.

 Front. Behav. Neurosci. 12:286. doi: 10.3389/fnbeh.2018.00286 Keywords: behavioral epigenetics, developmental science, methodology, study design, DNA methylation

### INTRODUCTION

Behavioral epigenetics (BE; Lester et al., 2011) refers to the study of epigenetic mechanisms and variations in association with exposures to early environmental conditions and phenotypic developmental outcome. The emergence of BE as a relevant field of research is due to the pioneering work by Meaney and Szyf (2005) who reported on epigenetic variations (i.e., DNA methylation) observed in rats exposed to varying levels of caregiving quality within a normal range. Notably, exposures to early environmental conditions might include both the experience of adversities (e.g., prenatal and post-natal stress exposures; Hyman, 2009) as well as protective care conditions (e.g., high-quality caregiving; Curley and Champagne, 2016). On the one hand, rats exposed to low quality of caregiving (i.e., low frequency of linking and grooming and arch-back nursing) showed high levels of methylation at the exon 1F of a specific stress-related gene (i.e., glucocorticoid gene, nr3c1), which in turn was predictive of developmental outcomes including behavior regulation, social interactions and stress reactivity (Turecki and Meaney, 2016). On the other hand, when rats born by mothers rated as low-quality caregivers were subsequently cross-fostered to mothers rated as high-quality caregivers their nr3c1 methylation patter was reversed to levels similar to those of rats born and raised by high-quality mothers and the detrimental effects on phenotype were no more observed (McGowan et al., 2011).

## THE EMERGENCE OF DEVELOPMENTAL HUMAN BEHAVIORAL EPIGENETICS

The fascination arising from these original animal model research intrigued researchers involved in human developmental science to test whether such epigenetic mechanisms might be involved in the processes through which early experience is embedded into the phenotype (Lester et al., 2016). The gene X environment approach was providing insightful support to the notion that—at some point in life—our genetic predisposition and the environmental encounters might interact resulting in observable behaviors (Belsky and Pluess, 2012; Mileva-Seitz et al., 2016). Nonetheless, BE holds promise to reveal the biochemical processes through which this interaction actually occurs in a developmental framework (Lester et al., 2011; Conradt, 2017).

Developmental human behavioral epigenetics (DHBE) studies—i.e., the application of BE to the study of human development—rapidly emerged in scientific literature. To date, this research has revealed that early experiences of prenatal stress (Oberlander et al., 2008; Devlin et al., 2010; Sosnowski et al., 2018), maltreatment and abuse (Beach et al., 2010; Blaze et al., 2015; Tyrka et al., 2016), neonatal pain (Provenzi et al., 2015; Montirosso et al., 2016a; Fumagalli et al., 2018), socio-economic disadvantage (Essex et al., 2013; Swartz et al., 2017) and other adversities might lead to altered patterns of DNA methylation that contribute to an individual's phenotype programming.

Nonetheless, very few DHBE studies to date focused on the epigenetic vestiges of protective care environmental conditions. For example, a retrospective study investigated the effect of maternal stroking on glucocorticoid receptor gene expression in infants that were exposure to maternal depression during pre and postnatal period (Murgatroyd et al., 2015). In this study, infants who were previously exposed to postnatal maternal depression showed a decrease of methylation after maternal stroking at 5 weeks of life. Consistent with this result, in a prospective study, the methylation correlates of physical maternal contact were measured in 4-to-5-year-old infants: those exposed to more frequent affectionate contact in the first 5 days of life showed greater methylation during preschool age (Moore et al., 2017).

## CHALLENGES IN DHBE RESEARCH

The misbalance between adversity- and care-focused BE research in human subjects is particularly evident. Different reasons might be invoked for such a predominant interest in adversity-related epigenetic variations in HBE. First, findings from animal models BE were more easily translated into study designs in human developmental science aimed at understanding the biological effects of early-in-life exposures to less-than-optimal caregiving contexts (Blaze et al., 2015). Second, the study of care-related epigenetic variations in animals can be relatively easy to set up with experimental manipulation, which is not the case of human subjects (Lester et al., 2011). Third, in humans protective and care interventions are delivered concurrently or—more often—after the occurrence of stressful, traumatic or adverse conditions and the timing of adversities and protective care is only partially predictable. As a result, in the absence of a conjoint and integrated DHBE research on both adverse and protective exposures, limited implications can be derived from this field of research that might have clear and insightful implications for clinical practice.

Moreover, the majority of the available DHBE research is characterized by retrospective study designs. The prevalence of correlational research adopting retrospective investigations might be at least partially related to the fact that when Meaney and Szyf's findings on rat's caregiving and DNA methylation variations began to be published, researchers involved in human studies started to assess DNA methylation by applying epigenetic investigation post hoc to ongoing cohorts. Another reason to set up more retrospective and cross-sectional studies than prospective and longitudinal ones in DHBE might be due to limitations of experimental manipulation in human subjects. As stressful or adverse conditions might not be ''administered'' to infants and children as well as to adult human subjects for ethical reasons, in most of the cases epigenetic mechanisms can be investigated only at post hoc. Notably, this limitation mostly applies to DHBE focused on early adversities, whereas it is less valid for BE research on the epigenetic correlates of protective care interventions in human subjects. Whereas retrospective and cross-sectional designs allow to set up DHBE research to reveal potential associations between environmental exposures and variations in DNA methylation, to date the lack of prospective and longitudinal research leads to reduced capacity of developing causal interpretations and theoretical modeling.

## THE PRESENT CONTRIBUTION

In the present study, we provide an in-depth discussion of different study designs in DHBE, we describe in details the advantages and limitations of different methodological architectures and we explore how different designs can be affected or solve major challenges in human BE research. When available, examples from research to date are also reported to better highlight the benefits and the challenges inherent to each specific methodological approach.

## RETROSPECTIVE AND CROSS-SECTIONAL STUDY DESIGNS

## Type 1: Concurrent Assessment of Epigenetics and Outcomes

#### Description

As reported in **Figure 1** (Type 1), this kind of study design implies the presence of an adverse exposure which has occurred before the study began (T0) and that can be only measured or accounted for through retrospective investigations such as self-report, medical charts, socio-demographic records and other

proxies. The core focus of this study design is on the assessment of both epigenetic variations and the identified outcome(s) at T1.

#### Example

In a recent article, Cicchetti and Handley (2017) reported on the retrospective investigation of the link between maltreatment, glucocorticoid receptor gene (i.e., NR3C1) methylation and several outcomes (i.e., emotional lability, self-control, behavioral problems, depressive symptoms) in a cohort of more than 500 school-aged children. Maltreatment information was obtained through the consultation of healthcare services records, classified in subtypes (i.e., neglect, emotional maltreatment, physical and sexual abuse) and characterized in terms of severity, age at exposure and chronicity. Cross-sectional assessment of NR3C1 methylation and outcomes occurred in a single research session. Children with history of early-onset maltreatment presented significant greater methylation of the NR3C1 gene when compared to non-maltreated counterparts. The cumulative exposure to more than one maltreatment subtype and to chronic maltreatment was significantly linked with NR3C1 hyper-methylation, which, in turn, was associated with greater emotional lability, lower self-control, more externalizing behavioral problems and depressive symptoms.

#### Advantages

The cross-sectional nature of the study is generally associated with a less expensive research in terms of human resources. Moreover, researchers do not have to wait for environmental conditions to develop in time in order to obtain assays of epigenetic biomarkers and outcomes, as the stressful conditions are already occurred prior to the study onset. Additionally, no experimental manipulations of environmental conditions are required, thus partially limiting the potentials of ethical issues for at-risk or fragile human subjects with a history of adverse exposures. This type of retrospective studies is well suited to investigate potential associations which have never been previously tested, providing preliminary evidence of putative significant correlations and co-variations between specific epigenetic markers and developmental outcomes.

#### Limitations

As the information on adversity exposure and stressful conditions can only be obtained indirectly, many efforts should be devoted in controlling as many confounding sources as possible. Nonetheless, the absence of direct account of stressful exposures (i.e., the actual duration or accurate observation of specific stress conditions) is a major flaw of this kind of DHBE study design. Additionally, the kind of depicted relationship among variables (i.e., retrospective adversity indexes, epigenetic markers and outcomes) can only be correlational and no assumption can be proposed on the direction of the effect between epigenetic marker and outcome measures. Finally, in absence of two sequential measures of epigenetic markers, there is no possibility to obtain a variability index assessing the actual effect of adversity on the quantitative change in the selected epigenetic marker.

## Type 2: Concurrent Assessment of Epigenetics, Care Exposures and Outcomes Assessment

#### Description

In this type of study design, adversity exposure already occurred before the subjects were enrolled in the research project (T0) whereas epigenetic markers, protective care conditions and outcomes are obtained within the same cross-sectional research session (T1; see **Figure 1**, Type 2). Compared to Type 1 study design, this methodological setting adds the integrated investigation of adversity and protective care exposures in a single DHBE research program.

#### Example

Conradt et al. (2016) investigated the association among the exposure to maternal post-natal depression, DNA methylation of NR3C1 and placental 11β-hydroxysteroid dehydrogenase Type 2 gene (i.e., 11β-HSD-2), salivary cortisol reactivity to socioemotional stress (i.e., the five-episode Face-to-Face Still-Face; Provenzi et al., 2016) and maternal sensitivity in 128 4-monthold infants. Retrospective self-report measure of maternal depressive symptoms was obtained through questionnaire administration. Infants' DNA methylation, hypothalamicpituitary-adrenal (HPA) axis regulation in response to the FFSF procedure and maternal sensitivity were all measured in the same research session at 4-month-age. As such, this study design is cross-sectional and retrospective, but has the merit of providing a preliminary account of the concurrent associations of adversity (i.e., maternal depression) and protective care (i.e., maternal sensitivity) with infants' DNA methylation and developmental outcome (i.e., stress regulation). Indeed, the authors showed that infants from depressed mothers who exhibited high levels of sensitivity during the face-to-face interaction occurring just before the still-face episode of the FFSF procedure had lower NR3C1 and 11β-HSD-2 methylation levels compared to counterparts from depressed mothers with lower sensitivity ratings. Consistent with previous animal model research (Curley et al., 2011), this study suggests that the quality of maternal caregiving behavior might be a significant protective buffer in the face of early adversity exposure in human infants.

#### Advantages

The limited impact on human resources and the possibility to obtain in relative short amount of time information on a whole cohort of subjects also applies to Type 2 study design. Also, limited manipulation of experimental conditions is allowed and care dimensions usually include naturally available protective factors such as socio-economical conditions or normal variations in parental care. These studies also have the advantage of testing uninvestigated hypotheses on the potential buffering or mediating role of protective care factors on the association among early adversities, epigenetic markers and selected developmental outcomes.

#### Limitations

Two major flaws affect this kind of study design: first, the indirect and retrospective account of adversities, as in Type 1 research; second, the concurrent assessment of protective factors and outcomes, which do not allow the interpretation of the direction of potentially significant associations. As such, while Type 2 research increases the complexity and provides more information compared to Type 1 study design, this DHBE research is still characterized as correlational and more careful interpretations of the study results should be done. Still, only epigenetic markers—but not variability indexes associated with adverse and protective exposures—are usually obtained in Type 2 research.

## Type 3: Retrospective Exposure to Adversity/Care and Concurrent Assessment of Epigenetics and Outcomes

#### Description

In this kind of study design, three time-points are considered (**Figure 1**, Type 3). Adversity exposure occurred in T0, usually well before (e.g., previous generation) the actual assessments included in the research program. Protective factors occurred in T1, which is still antecedent to the study onset, but subsequent to adversity in T0. The research assessments occur in T2 and usually include at least one of the following measurements: (a) epigenetic marker (e.g., DNA methylation) and (b) developmental outcome (e.g., healthrelated indexes). As consequence, in this kind of studies, both adverse and protective exposures are measured indirectly through retrospective investigations, whereas epigenetic and outcome measurements occur usually concurrently in one research session.

#### Example

The project Ice Storm is a well-suited example of the Type 3 study design (Cao-Lei et al., 2018). In this research, adversity (T0) consists in the well-known ice storm disaster that occurred in Quebec in 1998 and which offers a unique condition to assess intergenerational effects of early stressful conditions (King and Laplante, 2005). Ice storm exposure was quantified through retrospective questionnaire measuring the ''objective hardship'' of the exposure, including threat, loss, scope and change. Protective factor (T1) consisted in the maternal capacity of cognitive reappraisal coded as ''negative'' vs. ''neutral and positive'' meaning making and coping strategies. At the time of assessment (T2), enrolled subjects from the generation subsequent to that exposed to the 1998 ice storm averaged 13.6 years old and they were assessed for C-peptide secretion in blood T cells and for DNA methylation at selected genes associated with risk of Type 1 and 2 diabetes. The main merit of this study is the possibility to test for independent effects of adversity (i.e., objective hardship) and protection (i.e., maternal cognitive reappraisal) as well as for mediation models including both environmental exposures and epigenetic markers (i.e., DNA methylation at target sites within selected genes). Indeed, the authors showed that a direct marginal effect emerged only for objective hardship, whereas DNA methylation of diabetesrelated genes significantly mediated the effects of objective hardship (positive mediation) and cognitive reappraisal (negative mediation) on risk for diabetes (i.e., C-peptide secretion) in children.

#### Advantages

Although both adversities and protective care exposures are quantified at post hoc, this study design allow researchers to build a more complex model of the interactions as well as of the individual and joint contributions that environmental conditions exert on infants' epigenetic regulation and developmental outcomes. More specifically, the presence of a temporal sequence that organizes stress and protection conditions in the past of enrolled individuals adds to the possibility to develop a theoretical framework in which the buffering effect of protective factors can be appreciated.

#### Limitations

In the light of advantages listed above, major limitations still apply to this complex, yet retrospective study design. First, in the absence of an initial epigenetic assay at T0—or even before—there is no way researchers can observe and report on epigenetic regulation and variations in time and on the effects of environmental conditions for bad and for good on this time-related change. Second, despite a temporal sequence is hypothesized in the past occurrence of adversity and protective factors, careful interpretation of findings still applies to this kind of research, as it cannot be entirely excluded that other protective factors might have intervened concurrently or even before T0. Other flaws previously reported in relation to the retrospective assessment of adversity and protection (see Type 1 and Type 2 above) still apply to the present DHBE research program and only correlational interpretations of the relationship between epigenetic markers and developmental outcomes are sustained.

#### PROSPECTIVE AND LONGITUDINAL STUDY DESIGNS

## Type 4: Prospective Assessment of Adversity-Related Epigenetic Variations on Longitudinal Outcomes

#### Description

The key feature of this research architecture is the presence of at least two assessments of epigenetic markers at two different time points (see **Figure 2**, Type 4). Between the first (T0) and the second (T2) epigenetic assay, a prospective quantification of an adverse-related exposure is carried on (T1). A longitudinal assessment of a developmental outcome (T3) can be also included at least once after the second epigenetic assessment.

#### Example

Recent research applying epigenetic research principles and methodology to the study of early stress exposure in preterm infants during the hospitalization in the Neonatal Intensive Care Unit (NICU; Montirosso and Provenzi, 2015) represents a prototypic research context to describe Type 3 study design (Provenzi et al., 2017a). In this research program, epigenetic markers are obtained at birth (T0) and at NICU discharge (T2), whereas during the NICU stay (T1) specific variables related to stress exposures (e.g., pain-inducing skin-breaking procedures, mechanical ventilation, maternal separation) are quantified in a prospective way, usually on a day-byday basis. Outcomes (e.g., socio-emotional stress regulation; Montirosso et al., 2016a) are measured at specific post-discharge time-points (T3). In a recent article from this emerging field of research (Fumagalli et al., 2018), structural equation modeling was applied to assess the mediation role of epigenetic variation (e.g., difference in the methylation levels of the serotonin transporter gene, SLC6A4, from birth to discharge) on the association between NICU-related stress and anterior temporal lobes volumes at 12-month corrected age in preterm infants.

#### Advantages

The presence of two epigenetic measurements in different time-points allow researchers to move from epigenetic markers to epigenetic variations. Ratios and/or differential indexes of this variation in time can be obtained and are warranted to be tested in association with the target environmental exposure. Second, the kind of association that can be observed between adversity and epigenetic variation might also be characterized in terms of direction and hypothesis on the effect of adversity on epigenetic variation can be advanced. Third, a long-term chain of actionreaction sequences can be tested in complex models including all the variables, from the first epigenetic assay at T0 to the outcome assessment(s) at T3. More specifically, a prospective evaluation of the mediation of epigenetic variation on the association between adversity and outcome can be tested consistent with the temporal sequence in which environmental exposures and assessments occurred.

#### Limitations

Limitations of retrospective designs (Type 1–3) do not apply to prospective studies. Nonetheless, specific issues and challenges should be highlighted. First, these studies are much more expensive in terms of research procedures and human resources. Second, the longitudinal nature of the study exposes the research plan to moderate-to-high risk of sample attrition, as subjects enrolled at T0 might not be available for the subsequent research sessions from T1 to Tn due to many different reasons. Third, mainly for ethical reasons, contrary to what happens in animal model research, the adversity exposure cannot be experimentally manipulated or induced. As such, human populations who are naturally exposed to less-than-optimal developmental conditions featuring stress exposure are the best eligible subjects for this kind of research. In other words, this study design should be considered as quasi-experimental and careful interpretation of the direction of the effects is recommended.

## Type 5: Clinical Trials of Care-Related Epigenetic Variations on Longitudinal Outcomes

#### Description

This study design differs from Type 4 basically because of the type of environmental exposure which is assessed, namely protective care conditions (see **Figure 2**, Type 5). Indeed, between the first (T0) and the second (T2) epigenetic assay, some kind of protective care intervention is promoted and offered to at least one sub-group in the study sample (T1). A longitudinal assessment of a developmental outcome (T3) can be also included at least once after the second epigenetic assessment.

#### Example

Research on the effects of brief psychotherapy interventions appear to be among the best examples of the Type 5 prospective DHBE study design. Roberts and collaborators (Roberts et al., 2014) assessed SLC6A4 methylation before (T0) and after (T2) the exposure to a cognitive-behavior therapy (T1) in children with anxiety disorder. Post-treatment effects were measured in terms of a reduction of anxiety symptoms occurring at 6-month follow-up (T3). The findings suggested that SLC6A4 methylation partially explained the difference in the reduced anxiety symptomatology between children who improved and those who did not after the psychotherapy.

#### Advantages

design.

The inclusion of a protective care intervention instead of an adverse condition in T1 is not only a mere difference in terms of the object of investigation. Rather, the possibility to set up an intervention implies much more control for researchers and this kind of study design actually can be described as a clinical trial, with variable degrees of control and randomization. As such, this is probably one of the most robust methodological architectures for DHBE research. At the same time, this field of research holds potentials for the most direct implications and insights to advance evidence-based clinical practice and to inform early healthcare interventions for at-risk infants and children.

#### Limitations

The focus on prospective exposure and measurement of protective care interventions requires the setup of an integrated multi-professional research team and an accurate standardization of the intervention. At greater degrees of methodological control of confounders, more than one group of subjects is needed and infants/children included in the groups need to be matched for a number of socio-demographic, clinical and task-relevant variables. At lower degrees of control and randomization, only one group can be included, but less robust interpretations are supported.

## Type 6: Prospective Assessment of Adversity-Related Epigenetic Variations on Longitudinal Outcomes With Concurrent Exposure to Protective Care

#### Description

Many variants of Type 4 and 5 study designs can be developed to include an integrated assessment of both adversity and protective care exposures into a DHBE research project. One of this variant presents the inclusion of a concurrent assessment of a specific protective exposure at T3—together with the target outcome measurement—increasing the complexity of Type 4 study design (see **Figure 2**, Type 6).

#### Example

Preterm birth and its consequences in terms of early hospitalization and developmental outcomes represent an elite condition for designing Type 6 research projects. Using this kind of methodological architecture, Provenzi and colleagues (Provenzi et al., 2017b) assessed the moderation role of maternal sensitive interactive behavior during a face-toface interaction (T3) with full-term and preterm infants on the association between NICU-related (T1) variations in SLC6A4 methylation (T0 and T2) and infants' behavioral stress regulation (i.e., negative emotionality; T3) at 3 months of age (corrected for prematurity). Notably, this study suggested that a significant moderation emerged only for full-term infants, in which mothers rated as highly sensitive appeared to protect their infants from the positive association between high SLC6A4 methylation and heightened negative emotionality. By converse, no significant moderation emerged for preterm infants, indirectly supporting the need of DHBE research focused on an integrated investigation of NICU-related adversity and specific early interventions occurring well before outcomes' assessment (see Type 7 below).

#### Advantages

Type 6 study design combine the benefits of a prospective and longitudinal research project reported above (see Types 4 and 5) with the opportunity to appreciate the role of protective factors as post-adversity buffering mechanisms on developmental outcomes. This methodology is highly indicated and potentially insightful for areas of clinically applied research in which high individual variability is documented as a consequence of early stress exposure. In other words, this study can reveal the biological pathways that contribute to the fact that some individuals seem to cope adequately and develop well in the face of a developmental history of adversities, whereas others do not.

#### Limitations

Despite the many advantages of this study design, two major flaws should be highlighted. First, this kind of research can be applied only to those human developmental conditions that are inherently distressful and in which infants or children are naturally exposed to stressful environments (see Type 4 limitations). Second, the concurrent evaluation of outcomes and care variables do not allow researchers to assess the independent role of protective factors as contributors to epigenetic regulation.

## Type 7: Prospective and Sequential Assessment of Adversity- and Care-Related Epigenetic Variations on Longitudinal Outcomes

#### Description

This is probably the most complex study design within the landscape of DHBE research, as at least seven time-points are included and complex relationship among exposures, epigenetic variations and outcomes are addressed (see **Figure 2**, Type 7). Epigenetic assessments occur before (T0) and after (T2) an adverse event exposure (T1). After that, protective care intervention is promoted and quantified prospectively (T3) and a third epigenetic assessment occurs after that (T4). At least one post-intervention time-point is included for follow-up evaluation of the developmental outcomes (T5). Unfortunately, to the best of our knowledge, there is no evidence of such research projects in the available DHBE literature.

#### Advantages

The sequential order of exposures and epigenetic as well as outcome assessments allows researchers to develop specific and methodologically sound hypotheses that can tested and falsified with a modular approach. These can include simple hypotheses regarding two or three subsequent time-points as well as more complex modeling hypotheses including the integrated assessment of different environmental exposures, epigenetic variations and outcomes. The assessment of the competitive, additive or independent effects of adversity and protective care on the same targets of epigenetic variations and developmental outcomes is a specific plus of this study design which cannot be pursued with other methodological architectures.

#### Limitations

Whereas this study design maximizes the benefits at the methodological level, it should also be clear that resource costs, the risk for longitudinal sample attrition and the involvement of a multi-professional team of researchers and clinician imposes relevant challenges so that risks and potential solutions need to be carefully planned before and revised during the research project. At the same time, these research projects can be only applied to naturally occurring adversities for which protective care interventions are already available and provided some levels of evidence for the specific selected developmental outcomes. From this point of view, research on preterm behavioral epigenetics (PBE) still represents an elite field of applied research for DHBE (Maddalena, 2013; Provenzi and Barello, 2015). Nonetheless, it should be noted that a simplified version of Type 7 study design can be also suggested, in which a mixed retrospective assessment of adversity exposure and a prospective evaluation of care-related epigenetic variation together their effects on developmental outcomes of are featured. In this mixed design, T0-to-2 would be collapsed into one single retrospective assessment, reducing the interpretive potentials of the model, but allowing its application to many different adverse conditions and reducing the risk of longitudinal sample attrition.

### DISCUSSION

DHBE is one of the most promising and rapidly growing areas of developmental science in human psychobiological research field. These studies are well-recognized as holding promises of advancing our comprehension of the biological mechanisms underlying the association between environmental exposures and the developmental phenotype (Szyf et al., 2008; Griffiths and Hunter, 2014). Additionally, the growing body of information on the epigenetic targets and markers of early environmental exposures—for bad and for good—is going to inform smarter and effective evidence-based clinical practice for infants and children who present genetic and/or environmental risk factors (Bianco-Miotto et al., 2017; Murgatroyd and Spengler, 2011; Notterman and Mitchell, 2015). Nonetheless, DHBE presents specific challenges and issues, when it comes to make methodological choices and produce a robust study design. In the present contribution, we have proposed several different prototypical and didactically organized study designs to cope with these challenges.

One major distinction among different methodological approaches regards the use of retrospective or prospective measurements of environmental exposures, either adversities or protective conditions. Retrospective approaches clearly have the advantage of being applicable to a number of adverse conditions which can be faced by human beings and that cannot be experimentally induced or manipulated (e.g., abuse and maltreatment; Beach et al., 2013; Parent et al., 2017). Moreover, retrospective study designs represent a compromise solution that balances the amount of resources needed to the research project and the strength of methodological procedures and robustness of findings. Indeed, retrospective designs are usually correlational and no reliable interpretation can be supported for what pertains predictive effects and the direction of potential significant associations. As such, these study designs are better suited for exploratory research projects and to test innovative and previously uninvestigated hypotheses that hold the potential to pave the way for more research in the field.

By converse, prospective approaches usually implies more complex study designs which increase the robustness of findings' interpretation, allow the formulation of more specific hypotheses on the direction of predictive associations and effects. At the same time, they imply new challenges such as the high risk of sample attrition and subjects' loss and the careful planning of multiple research sessions within specific time frames. Prospective study designs are highly indicated when environmental conditions—for bad and for good—can be longitudinally followed-up, monitored and quantified and/or when greater opportunities for experimental manipulation and control is granted. For instance, this is the case of PBE research (Provenzi et al., 2018a), in which the early exposure to adverse conditions (e.g., NICU-related pain-inducing procedures) and care interventions (e.g., skinto-skin contact support) are expected and can be easily measured on a daily basis for a relatively long period (Provenzi et al., 2015; Montirosso et al., 2016a). Prospective DHBE studies are particularly well suited for the investigation of specific hypotheses regarding the predictive effects of adversities and protective care interventions on target epigenetic regulation.

Another major difference between retrospective and prospective study designs regards the kind of epigenetic indexes that can be obtained. On the one hand, retrospective and cross-sectional study designs provide the context for the assay of punctual epigenetic markers, which can be considered as potential endophenotype associated with specific environmental exposures (Radley et al., 2011). On the other hand, prospective research allows the measurements of epigenetic variations from pre- to post-exposure, which further increases the possibility to provide stronger interpretations on the direction of the effects and supports the development of more complex mapping of the reciprocal interactions among environmental conditions, epigenetic regulation and developmental outcomes. In other words, while retrospective approaches to DHBE provide photograph-like accounts of the relationships among environmental exposures, epigenetic markers and individuals' phenotype, prospective study designs provide the methodological requisites to depict a movie-like systematic landscape of such relationships.

In light of considerations reported above, we would like to highlight specific implications for future studies in DHBE research field. First, a more explicit account of the limitations of the adopted study design and their advantages and disadvantages for direct and indirect implications and interpretations should be provided to readers in DHBE studies. As previously suggested, the ''seductive allure'' (Miller, 2010) of BE research and its application to human developmental science might pave the way for misinterpretations in the absence of scientifically sound supervised and guided readership (Richardson et al., 2014; Provenzi and Montirosso, 2015). Second, as more and more DHBE research get published in the scientific community, there is a need to provide systematic accounts of this rapidly growing body of evidence, to weight them and to highlight the gray areas of under-investigated yet clinically relevant areas of BE applications for human developmental research. For example, the imbalance between DHBE research focused on adversities and on protective care exposures is evident and more efforts should be devoted to care-related DHBE studies in the years to come. Third, in the face of the described challenges inherent to the application of BE research principles to the study of human development, more direct and frequent contacts between animal model and human epigenetics research should be pursued. Investing in this multi-professional communities of BE researchers is warranted to provide a more systematic account and robust rationale for the applied and basic evidence that can support our improved knowledge of developmental mechanisms and our capacity to provide smarter and more effective early interventions in at-risk conditions. Additionally, it should be highlighted that the use of peripheral tissues for measuring DNA methylation variations is the only available proxy in DHBE, whereas epigenetic research on animal models rely on central nervous system tissues. Recent research reported on partial correspondence in DNA methylation measured from peripheral blood and saliva (Thompson et al., 2013; Staunstrup et al., 2017), but still methylation status appears to be tissue- (Smith et al., 2015; Forest et al., 2018) and gene-specific (Di Sante et al., 2018). Also, Walton et al. (2016) reported only 7.9% significant correlation between methylation measures in blood and brain tissue obtained in post-mortem patients: despite this proportion was greater than predicted by chance, it is still a small proportion. As such, a direct translation of findings from animal model studies to humans is discouraged, in absence of multiple evidence and cross-tissue reports (Lester et al., 2011). Finally, as previous DHBE research mainly focused on DNA methylation, in this article we limited our discussion and examples to this epigenetic mechanism. Nonetheless, it should be noted that DNA methylation can have direct and indirect effects of the transcription machinery (Curradi et al., 2002). Moreover, other epigenetic mechanisms have been documented in associations with early adversities, at least in animal models (e.g., histone regulation, Cittaro et al., 2016; non-coding RNA, Daskalakis et al., 2018; telomere length regulation, Provenzi et al., 2018b) and might interact with each other in contributing to establish chromatin availability to transcription and gene silencing (O'Leary et al., 2017).

In sum, it should be highlighted that when it comes to apply BE principles to human developmental science, there is no ''best approach'' and that the balance between benefits and challenges should be considered for each specific research project based on a conjoint evaluation of researchers' aims, objects of investigation and available resources. Although nonexhaustive, this article provides a wide overview of study designs and offers methodological support and guidelines to researchers who are interested in integrating BE methods and investigation in their developmental science research. A well-designed research program is key to develop robust

#### REFERENCES

methodology for future DHBE studies that can be beneficial for increasing and deepening our knowledge of developmental mechanisms and inform early interventions for at-risk infants and children.

#### AUTHOR CONTRIBUTIONS

LP conceived the idea. LP and MB drafted the manuscript. RM and RB critically revised the manuscript for important intellectual content. All authors gave final approval of the final version and are accountable for this work. The corresponding author attests that all listed authors met authorship criteria and that no others meeting the criteria have been omitted.

### FUNDING

This work was supported by a fund to author RB from the Italian Ministry of Health, Ricerca Corrente RC 01-05 2015-2018: ''Effetti a lungo termine di esperienze avverse precoci in bambini a rischio evolutivo Metilazione del DNA e lunghezza dei telomeri come biomarker di stress neonatale alla nascita e in età prescolare in bambini nati pretermine''.

### ACKNOWLEDGMENTS

We are thankful to colleagues from the 0-3 Center for the at-Risk Infant at the Scientific Institute IRCCS Eugenio Medea: Lorenzo Giusti, Giunia Scotto di Minico and Eleonora Visintin.

panic disorder: role for Asic1 and neurodevelopmentla genes. Sci. Rep. 6:25131. doi: 10.1038/srep25131

neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses. Epigenetics 3, 97–106. doi: 10.4161/epi.3. 2.6034

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Provenzi, Brambilla, Borgatti and Montirosso. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Socio-Emotional and Cognitive Development in Intrauterine Growth Restricted (IUGR) and Typical Development Infants: Early Interactive Patterns and Underlying Neural Correlates. Rationale and Methods of the Study

Chiara Sacchi 1 \*, Pietro De Carli 1 , Giovanni Mento1,2 , Teresa Farroni 1 , Silvia Visentin3 and Alessandra Simonelli 1 \*

1Department of Developmental Psychology and Socialization, University of Padova, Padova, Italy, 2Department of General Psychology, University of Padova, Padova, Italy, 3Department of Women's and Child's Health, University of Padova, Padova, Italy

#### Edited by:

Rosario Montirosso, Eugenio Medea (IRCCS), Italy

#### Reviewed by:

Claudio Lucchiari, University of Milan, Italy Anna Brancato, Università degli Studi di Palermo, Italy

\*Correspondence:

Chiara Sacchi<EMAIL_ADDRESS>Alessandra Simonelli<EMAIL_ADDRESS>
Received: 21 September 2018 Accepted: 03 December 2018 Published: 18 December 2018

#### Citation:

Sacchi C, De Carli P, Mento G, Farroni T, Visentin S and Simonelli A (2018) Socio-Emotional and Cognitive Development in Intrauterine Growth Restricted (IUGR) and Typical Development Infants: Early Interactive Patterns and Underlying Neural Correlates. Rationale and Methods of the Study. Front. Behav. Neurosci. 12:315. doi: 10.3389/fnbeh.2018.00315 Intrauterine growth restriction (IUGR) is defined as a fetal growth retardation, resulting in an estimated fetal weight less than the 10th centile for gestational age. IUGR developing brain is affected by the atypical fetal growth, presenting altered structure and connectivity and increased risk for neurodevelopmental impairments. Behaviorally, IUGR infants show reduced responsiveness and engagement with human faces during mother-child exchanges. The neural mechanisms of these patterns of interactions remain unexplored, as well as their potential role in shaping socio-cognitive trajectories of development. Aim of this research project will be to longitudinally investigate mother-infant interactions and infant's event-related potential (ERP) components of face processing (infant N170, P400, Negative central) in 4 and 9 months IUGR as potential early markers of expected atypical cognitive and behavioral outcomes observed at 12 months. Thirty IUGR participants will be recruited after receiving the in utero diagnosis (>28th gestational week). Thirty healthy infants will be enrolled as the control group. Maternal environment will be assessed via Emotional Availability Scales (EASs), with child responsiveness and maternal sensitivity as variables of interest. Infants' scalprecorded cortical activity in response to social and non-social stimuli will be investigated using a high-density EEG system (EGI Geodesic system). Neurodevelopment will be measured at 12 months of child's life, using Bayley Scales for Infant Development (BSID), while the possible presence of emotional-behavioral problems will be rated via Child Behavior Checklist (CBCL). We expect that being IUGR significantly affects cognitive and behavioral outcomes, through mediation effects of both infants' neural and behavioral capacity to respond to social stimuli. Indeed, we expect an altered response to social stimuli in IUGR infants, resulting in smaller ERP components amplitude in response to human faces compared to healthy matched peers. A significant association between neural response to social stimuli and infants' responsiveness to maternal stimulation during interactions is expected, with impoverished performances on the interactive domain in IUGR, compared to healthy peers. This study will enhance understanding on neural mechanisms underpinning the interactive patterns sustaining socio-cognitive development in IUGR and healthy infants. The study will help in clarifying the role of postnatal environment in buffering the vulnerability experienced by children delayed in their fetal growth.

Keywords: intrauterine stress, fetal growth restriction, face processing, socio-cognitive development, motherchild interactions

## INTRODUCTION

Intrauterine growth restriction (IUGR) is defined as a fetal growth retardation, resulting in an estimated fetal weight (postnatally confirmed by birth weight) on the lowest 10th percentile for gestational age (Alfirevic and Neilson, 1993). By affecting 5% to 7% of pregnancies, IUGR is the second leading cause of perinatal mortality and morbidity worldwide, representing a major public health problem (Murray et al., 2015). A fetal growth unable to reach its genetic potential is a risk factor for later neurodevelopmental outcomes (Kok et al., 2007; Baschat, 2011). In fact, functional impairments have been observed at birth and early in life; specifically, immature attention-interaction scores and impaired visual recognition memory performances are described in 7-month-old infants (Gotlieb et al., 1988; Tolsa et al., 2004), while at 1 year of life, significantly lower scores on Bayley Scales are reported (Fernandez-Carrocera et al., 2003; Batalle et al., 2013). Moreover, growth-restricted infants show poor use of environmental stimuli, reduced social responsiveness, more insulated cry states, and poor motor performance as compared with normal birth weight infants (Padilla et al., 2011). Persisting and long-term outcomes are also observed, with cognitive impairments (e.g., executive functioning; Geva et al., 2006) and behavioral problems described in childhood (Sung et al., 1993); motor problems, learning difficulties and lower academic achievements during school age period (Leitner et al., 2007; Esteban et al., 2010), as well as increased risk for neurodevelopmental disorders, such as ADHD (Heinonen et al., 2013). Apart from evidence of neurodevelopmental and cognitive outcomes, socio-emotional development still appears as unexplored in the developmental context of growth restriction, although few signs of early atypical social interactions are described (Watt, 1990; Feldman and Eidelman, 2009), as well as later poor socio-cognitive performances at school age and mood disorders (Fischi-Gómez et al., 2015).

Literature evidenced several structural and functional brain abnormalities potentially linking fetal growth rate to the detrimental neurodevelopmental and socio-cognitive outcomes. Indeed, IUGR infants show reduced brain volumes as well as delayed and diminished myelination (Dubois et al., 2008; Padilla et al., 2011; Ramenghi et al., 2011). The alterations seem to persist in long term deficits, since motor and cortico-striatal-thalamic networks impairments are observed in 6 years old IUGR children, and delayed myelination as well as disrupted white matter integrity last up to adulthood (Fischi-Gómez et al., 2015). Despite this evidence, the early neural mechanisms sustaining the socio-emotional competencies and the socio-cognitive development in IUGR infants are still underexplored. However, it is of the highest importance to provide comprehension on early markers, both in terms of behavioral and brain mechanisms, of the developmental cascade that begins with early fetal abnormal experience and might potentially result in socio-emotional difficulties and neurodevelopmental outcomes observed later in life. Indeed, in a preventive perspective, targeting a potential early vulnerability in IUGR development, particularly when born at term, could be highly convenient and rewarding. Despite literature extensively reports altered quality in antenatal environment, quite few studies investigated IUGR developmental trajectories, thus neglecting the opportunity to tailor interventions and to develop ad hoc follow up mental health care. In addition, the urgency for identifying potential targets for interventions should consider a multifaceted approach, where infant and caregiver are parts of a mutually influencing complex and interrelated system (Sacchi et al., 2018). Taking into account infant and mother's variables, different potential mechanisms to target could arise from this study. First, detecting vulnerability in processing social stimuli might guide behavioral intervention sustaining parenting abilities to use multi-modal channels of stimulations during interactions. Second, fostering a protective effect of parenting behavior on brain functionality would potentially have an effect on infant socio-emotional development, hopefully compensating for the suspected reduced early communication abilities of IUGR infants. Third, a longitudinal investigation would allow to study different potential windows of plasticity both for typical and atypical development. This could lead to more focused interventions aware of the most susceptible periods and the most rewarding processes to target.

With this theoretical and clinical perspective in mind, we propose a longitudinal investigation of two interrelated mechanisms that might be detected across the first year of life as early markers of potential atypical socio-emotional and cognitive outcomes of IUGR developmental trajectories: infant behavioral responsiveness in social interaction and infant early neural face processing.

## Infant Behavioral Responsiveness in Social Interaction

Within the first year of life, typically developing infants display an amazingly sophisticated set of social behaviors, which foster learning processes in a broad collection of developmental domains (McDonald and Perdue, 2018). These socio-emotional competencies involve the abilities to interact, communicate and deal with emotions, which are primarily experienced in early interactive exchanges with the mother (Bowlby, 1978). Within this affectionate bond, the child receives not only protection, care and the recognition of his/her needs, but also an encompassing environment for physical, cognitive, social and affective development (Britto et al., 2017). Indeed, the mutuality of exchanges between mother and child represents not only a source of stimuli for the child but also an environment sensitive to activities and modifications (van den Bloom and Hoeksma, 1994). In the case of atypical development, infant characteristics can deeply expose the quality of mother child interactions (Kiff et al., 2011). Indeed, the few available studies evidenced that IUGR infants are likely to display difficulties in orientating to social and non-social environment (Watt, 1990) and tend to look at people less frequently than age-matched healthy infants; also, higher levels of negative affect are reported, evidencing an early vulnerability in communication skills (Watt and Strongman, 1985; Watt, 1987). As regards, interactive abilities, the very limited findings on IUGR or small for gestational age (SGA) children suggest those infants are more passive during mother-child interactive exchanges, smiling and looking at their mothers' face less than normal birth weight matched newborns, being less rhythmic and synchronous in daily interactions (Feldman and Eidelman, 2006), and thus appearing as less rewarding interactive partners. A similar interactive pattern is displayed in preterm infants and their mothers, where a scarcity of communicative signals on infant's side could activate some compensatory behaviors in parents (Miles and Holditch-Davis, 1995; Montirosso et al., 2017). This parenting response can be highly adaptive but can eventually result into intrusive and non-attuned behaviors (Howe et al., 2016). Therefore, on the one hand, atypical development in the domain of diminished early communicative abilities can disrupt mother child exchanges leading to an additional impoverishment of infant's environment. On the other hand, the quality of mother-child interactions can potentially buffer the negative effect of infant scarce interactive abilities on child development (Baker et al., 2007). In fact, some evidences show the moderating role of maternal sensitivity on infant developmental trajectories. More specifically, recent evidences show the role of mother child interactions on infant's brain functionality, confirming the relevance of considering mother and child as a broad interrelated system where infant development takes place.

### Infant Neural Face Processing

Among early neural competences displayed by newborn and infants, the ability to recognize and direct the attention to faces appear as highly relevant for socio-cognitive development. Face perception represents an experience-expectant and activitydependent function (Nelson, 2001; Young et al., 2017); that is critical in the development of higher level social and cognitive functions (Parker and Nelson, 2005). Indeed, the human face provides the infant with a wealth of socially and affectively relevant information and humans appear to be inherently interested in faces, displaying from infancy a strong interest in facial-like figures (Johnson, 1991; Morton and Johnson, 1991). Early disruption or delay in this low-level process can negatively impact infant's ability to interact with the social environment (Elsabbagh et al., 2015) and disturb natural mutuality in social interaction with potential detrimental effects for child development (Wan et al., 2013). Indeed, huge part of early interactive exchanges rely on the use of face and facial expression are early used to understand others emotion and thought, to make others understand themselves, and to share emotional states (Beebe et al., 2010, 2016). Studies observed that different early stressors and risk factors, such as prolonged institutionalization or risk for autism, are likely to affect this infant capacity that is considered a strong candidate for being one of the mechanisms of the association between early stress and socio-emotional difficulties (Nelson and McCleery, 2008). More specifically, Parker and Nelson (2005) found that the amplitude of the event-related potential (ERP) responses to familiar and unknown faces were lower in institutionalized children, while Swingler et al. (2010) found ERP latencies to be associated with infant behavioral response to maternal separation. Mesquita et al. (2015) showed altered ERP components magnitude in response to faces in children with atypical social behaviors and recently (Kungl et al., 2017) found an association between attachment security and face brain responses. In addition, recent studies show that in healthy children the quality of the maternal environment is related to the magnitude of ERP components in response to emotional faces (Carlsson et al., 2008; Taylor-Colls and Pasco Fearon, 2015), confirming the association between early interactive experience and brain development of face perception. As a consequence, it is possible that an early dysfunction of the relevant circuitry of neural face processing could affect the quality of the interactions, and probably also decrease the quality of the child environment, contributing to activate a negative developmental pathway. Up to date limited information is available on how infants process and respond to social stimuli in early at-risk conditions. In particular, no study investigated whether early human face processing is susceptible to antenatal growth and/or might be affected by fetal growth restriction. Indeed, in the study of IUGR, researches are needed in order to ensure that facial processing is not compromised by their antenatal adversities slowing down the fetal growth. In fact, in the light of studies on clinical groups (Parker and Nelson, 2005; Nelson and McCleery, 2008), it appears as of highest clinical importance to understand the role of early adversities on neural face processing and how altered face processing could be conceived as early marched on possible risk on socio-emotional development.

With the aim of bridging the above-described research focuses and objectives, the present study protocol attempts to open a new research perspective on early development of IUGR infants, following their interactive and neural developmental pathways across the first year of life. By comparing IUGR with healthy children, we study the effect of antenatal adversity on brain functionality and interactive abilities. Specifically, aim of the study will be to investigate whether growth restriction significantly affect socio-cognitive developmental at 12 months both directly and thought the mediation of behavioral and neural response to social stimuli as displayed at 4 and 9 months. In particular, mediation hypotheses cover the following pathways:

Then, cortical response to social stimuli will be investigated as possible underpinnings of reduced responsiveness to maternal environment in IUGR infants, compared to matched healthy controls. Therefore, positive correlations will be expected, highlighting this link.

Last, along with the mediation roles expected for child behavioral and neural social responsivity, maternal environment, in terms of sensitivity, will be investigated at an explorative level as exerting a moderating role in the association between infant responsivity on cognitive and behavioral development assessed at 12th months. Indeed, although no specific evidence suggests that maternal sensitivity can modulate the developmental trajectories in IUGR samples, this effect has been shown in other atrisk population such as premature infants. Therefore, we aim at considering moderator effects in order to detect potential buffering or detrimental roles of maternal environment in infants experiencing fetal stress.

Overall, research design and hypotheses are graphically summarized in **Figure 1**.

## METHOD

## Participants

For the IUGR group, 30 pregnant women will be recruited at the Department of Women's and Child's Health, University of Padova (Italy). Healthy control pregnant mothers (N = 30) will be recruited from birth-preparation courses of the Obstetrics and Gynecological Clinic of Padua Hospital. All pregnant women are orally presented with a longitudinal study on the role of IUGR on child socio-emotional development by a Gynecologist and a Psychologist, while waiting their visit or the birth-preparation class. For mothers with pregnancy complicated by IUGR; research will be proposed at the first obstetrical visit following diagnosis. All mothers interested in the study will receive a detailed informative module, describing stages and tasks on the study. In particular, they will be informed on the study length and procedures; absence of risk for both behavioral and EEG assessment is declared, and a potential tolerable level of discomfort is reported for the EEG cup wearing procedure. Participants will also be informed on the possibility to withdraw their participation at any time without giving an explanation and that their decision would not affect future healthcare encounters. In accordance with the Declaration of Helsinki, prior to first assessment, parents agreeing to be involved in the present study will sign written informed consent. Mothers will sign an informed consent as participants; while two other different consent forms are required to be signed by both parents for infant's participation, namely in the behavioral and neuroimaging assessments. The present study received ethical approval from the Ethic Committee of the University of Padua (protocol reference number: 2293).

Participants will be mother-infant dyads who received in utero IUGR diagnosis (verified by Doppler ultrasound and estimated birth weight below the 10th percentile of growth), confirmed by birth weight below the 10th percentile of growth curve. Infants exclusion criteria will be: genetic disorders, unrelated comorbidities, presence of fetal infections, congenital malformations (i.e., congenital heart disease), metabolic and chromosomal disorders at birth, as well

as infant neurological pathologies, brain abnormalities, or preterm delivery (<37th gestational week). Mothers' exclusion criteria will be IUGR diagnosis before the 7th month of pregnancy and complicated pregnancies, non-Italian nationality, mother age <18 years, psychiatric disorders' risk as defined by clinical score (namely a Global Symptom Index >65) in the Symptom Checklist 90-Revised (Derogatis, 1975), neurocognitive disorders, drug addiction, single mothers.

## Procedure

This project describes a longitudinal research, articulated in four stages over the first year of the child's life. During recruitment at the pregnancy stage demographical information will be collected through a detailed paper-and-pencil evaluation. Indeed, an ad hoc socio-demographic assessment has been designed in order to collect comprehensive information about maternal age, cohabitation, marital status, education, work, parity and presence of previous abortion or at-risk pregnancies.

In addition, psycho-social and clinical-psychological status of the mother and of the whole family system will be assessed, by applying the following self-report questionnaires: Childhood Trauma Questionnaire (CTQ)—Short Form (Bernstein et al., 2003; Sacchi et al., 2017); Parental Bonding Instrument (PBI; Parker, 1989); Difficulties in Emotion Regulation Scale (DERS; Gratz and Roemer, 2004); State Trait Anxiety Inventory (STAI; Spielberger, 1983); Beck Depression Inventory-II (BDI-II Beck et al., 1996).

After recruitment and the first assessment during pregnancy taking place at the Hospital, all participants agreeing to take part into the study will be telephonically contacted at the 4th month of child's life for behavioral and neuroimaging assessment. In particular, mother-child couples will be invited to visit the Department of Developmental Psychology and Socialization at the University of Padova. At 4 and 9 months, assessment procedure will involve free play interactions and EEG recording at the Inter-departmental High-density EEG lab; while at 12 months, developmental outcomes will be measured with children assessed on cognitive development by a structured procedure performed by trained psychologist, and emotionalbehavioral problems rated by mothers.

#### Cognitive and Socio-Emotional Development at 12 Months of Life

Cognitive assessment will be performed at 12 months of child's life using Bayley Scales for Infant Development—Third Version (BSID—III; Bayley, 2006); which evaluates five different domains: cognitive, language, motor, socio-emotional behavior and adaptive behavior. Evaluation of the first three domains consist of a direct observation of the child performance on different task, while socio-emotional and adaptive behaviors are parent rated. For direct assessment, each item is assessed on a dichotomous scale, with 1 given to child's ability to perform the targeted behavior and 0 to the absence of such behavior. After five consecutive missing behaviors the scale's administration is interrupted. Cognitive scale is composed by 91 items assessing: sensorimotor development, exploration and manipulation, object relatedness, concept formation, and memory. Language scale is composed by 49 items referring to receptive communication (i.e., pre-verbal behavior, vocabulary development, morphological development, understanding morphological markers, social referencing and verbal comprehension), and 48 items assessing pre-verbal communications (i.e., vocabulary development and morphosyntactic development). Motor scale examines fine motor and gross motor domains. In particular, fine motor subtest is composed by 66 items about: prehension, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, responses to tactile information. Gross motor subtest refers to 72 items covering movement of the limbs and torso, static positioning (e.g., sitting, standing), dynamic movement (including locomotion and coordination), balance, and motor planning.

Socio-emotional scale represents and adaptation from the Greenspan social-emotional growth chart (Greenspan, 2004) assessing child self-regulation, communicating needs, the ability to establish relationship and the use of emotions for interactive purposes or to solve problems. Last, Adaptive Behavior assessment refers to child's social, motor, pre-academics, home living, self-care, self-direction, community use, leisure, communication, health and safety skills.