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Effects of OXTR on maternal depressions and dyadic emotional availability

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Title:
Effects of OXTR on maternal depressions and dyadic emotional availability
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Asherin, Ryan M. ( author )
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Denver, Colo.
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Doctorate ( Doctor of philosophy)
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University of Colorado Denver
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Department of Psychology, CU Denver
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Clinical health psychology

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Depression in women ( lcsh )
Mothers ( lcsh )
Oxytocin -- Research ( lcsh )
Depression in women ( fast )
Mothers ( fast )
Oxytocin -- Research ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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BACKGROUND: Recent research has demonstrated a link between polymorphisms in the oxytocin receptor gene (OXTR) and increased psychopathology, non-optimal parenting behaviors, and negative outcomes in child development. Genotypic polymorphism of OXTR is expressed through the homozygous or heterozygous pairing of alleles A and G. Comparisons of individuals with different OXTR genotypes have suggested that adult carriers of the A allele display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, poorer parenting skills, decreased empathy, and worse mental health outcomes compared to homozygous G/G adults (Riem, Pieper, Out, Bakermans-Kranenburg, & Van Ijzendoorn, 2011). However, despite the reported benefits to parenting behavior associated with the homozygous G/G OXTR genotype, it has also been associated with a greater risk of developing major depression (R.J. Thompson, Parker, Hallmayer, Waugh, & Gotlib, 2011). In addition, given the bidirectional influences of parent and child in both caregiver sensitivity and maternal depression (Feldman, 2012b, 2017), an important unaddressed question concerns effects associated with the child's genotype or the concordance/discordance between dyadic genotypic variations. The objective of this dissertation was to examine relationships that may exist between mothers and their infants in relation to oxytocin receptor types, maternal depression, and mother and child dyadic emotional availability, as measured by the empirically supported Emotional Availability Scales (EAS; Biringen, 2008). Four specific hypotheses were tested: (1) that the presence of depression in the postpartum period will be related to the presence of the G/G OXTR allele sequence, (2) that mothers carrying an A OXTR allele will demonstrate decreased levels of Sensitivity as measured by the EAS, (3) that infants of depressed mothers who are carriers of an A OXTR allele will be protected from some of the adverse effects of maternal depression and demonstrate reduced dyadic sensitivity, and (4) that non-depressed mother-child dyads that are both carriers of the homozygous G genotype will demonstrate significantly higher dyadic emotional availability ratings. In addition, although specific a priori predictions were not made, analyses of the unique contributions of infant OXTR genotype and mother-infant concordance/discordance on maternal functioning and mother-infant interactions were carried out in posthoc analysis. METHODS: Mother-infant dyads (N=61) were recruited using an advertisement posted on Facebook. Mothers were asked to complete demographic questionnaires, several self-report depression inventories, a Structured Clinical Interview for DSM-IV Axis I Disorders (SCID), and a 10-minute semi-structured play session for later coding using the EAS. Infant cognitive and communicative development were assessed using the Bayley Scales of Infant and Toddler Development (3rd Edition; BSID-II). Salivary samples were obtained via buccal swab for later genotypic analyses. DNA isolation was preformed according to manufacture specifications (Isohelix DNA Isolation Kits: DDK-3/DDK-50) before genetic sequencing of OXTR rs53576 was completed through the use of a STEPONE qPCR machine. CONCLUSIONS: Results of this study suggest that maternal genotype alone may not be enough to discriminate predictors of complex dyadic interactions. Biobehavioral synchrony between mother and infant, and specifically OXTR genotype concordance/discordance, may offer increased insight into the role oxytocin and OXTR play in observable dyadic emotional availability, as well as the greater development of pair bonding, attachment, and social affiliation. Contrary to expectations from the literature, mothers with the GG OXTR genotype were not more sensitive or more prone to depression. If anything, this genotype was related to greater maternal intrusiveness during a semi-structured play interaction. However, GG mothers showed better sensitivity if their infants were GG than AG/AA, suggesting either that concordance promotes optimal biobehavioral synchrony and functioning of the oxytocin system or is a proxy for some other, unmeasured, variable that leads to better dyadic interactions. Possible mechanisms to explain the association between infant OXTR genotype and maternal depression, and between OXTR concordance and maternal emotional availability, are discussed. ( ,, )
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by Ryan M. Asherin.

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Full Text
EFFECTS OF OXTR ON MATERNAL DEPRESSION
AND DYADIC EMOTIONAL AVAILABILITY
by
RYAN M. ASHERIN
B.A., Metropolitan State University of Denver, 2004 M.A., University of Colorado Denver, 2014
A dissertation submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Clinical Health Psychology Program
2017


This dissertation for the Doctor of Philosophy degree by Ryan M. Asherin has been approved for the Clinical Health Psychology Program by
Peter Kaplan, Chair Kevin Everhart, Advisor Dave Albeck Jim Grigsby
Date: May 13, 2017
11


Asherin, Ryan M. (Ph.D., Clinical Health Psychology Program)
Effects of OXTR on Maternal Depression and Dyadic Emotional Availability Dissertation directed by Professor Kevin Everhart.
ABSTRACT
Recent research has demonstrated a link between polymorphisms in the oxytocin receptor gene (OXTR) and increased psychopathology, non-optimal parenting behaviors, and negative outcomes in child development. Genotypic polymorphism of OXTR is expressed through the homozygous or heterozygous pairing of alleles A and G. Comparisons of individuals with different OXTR genotypes have suggested that adult carriers of the A allele display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, poorer parenting skills, decreased empathy, and worse mental health outcomes compared to homozygous G/G adults (Riem, Pieper, Out, Bakermans-Kranenburg, & Van Ijzendoorn, 2011). However, despite the reported benefits to parenting behavior associated with the homozygous G/G OXTR genotype, it has also been associated with a greater risk of developing major depression (R. J. Thompson, Parker, Hallmayer, Waugh, & Gotlib, 2011). In addition, given the bidirectional influences of parent and child in both caregiver sensitivity and maternal depression (Feldman, 2012b, 2017), an important unaddressed question concerns effects associated with the childs genotype or the concordance/discordance between dyadic genotypic variations. The objective of this dissertation was to examine relationships that may exist between mothers and their infants in relation to oxytocin receptor types, maternal depression, and mother and child dyadic emotional availability, as measured by the empirically supported Emotional Availability Scales (EAS; Biringen, 2008). Four specific hypotheses were tested: (1) that the presence of depression in the postpartum period


will be related to the presence of the G/G OXTR allele sequence, (2) that mothers carrying an A OXTR allele will demonstrate decreased levels of Sensitivity as measured by the EAS, (3) that infants of depressed mothers who are carriers of an A OXTR allele will be protected from some of the adverse effects of maternal depression and demonstrate reduced dyadic sensitivity, and (4) that non-depressed mother-child dyads that are both carriers of the homozygous G genotype will demonstrate significantly higher dyadic emotional availability ratings. In addition, although specific a priori predictions were not made, analyses of the unique contributions of infant OXTR genotype and mother-infant concordance/discordance on maternal functioning and mother-infant interactions were carried out in posthoc analysis.
Mother-infant dyads (N=61) were recruited using an advertisement posted on Facebook. Mothers were asked to complete demographic questionnaires, several self-report depression inventories, a Structured Clinical Interview for DSM-IV Axis I Disorders (SCID), and a 10-minute semi-structured play session for later coding using the EAS. Infant cognitive and communicative development were assessed using the Bayley Scales of Infant and Toddler Development (3rd Edition; BSID-II). Salivary samples were obtained via buccal swab for later genotypic analyses. DNA isolation was preformed according to manufacture specifications (Isohelix DNA Isolation Kits: DDK-3/DDK-50) before genetic sequencing of OXTR rs53576 was completed through the use of a STEPONE qPCR machine.
Contrary to predictions, maternal OXTR genotype was not associated with increased rates of maternal depression, as measured either by common PPD screening measures or via structured clinical interview. Variations in maternal OXTR genotype alone did not account for a significant difference in global ratings of maternal Sensitivity or Child Responsiveness on the EAS, nor did it account for child BSID-III performance. However, both maternal and
IV


child OXTR genotype were significantly association with maternal non-intrusiveness, with the GG genotype in both cases linked to greater maternal intrusiveness. Furthermore, infant OXTR genotype was linked to significant differences in DSM-IV Axis-I depression-spectrum diagnoses of the mother, as well as to the mothers interviewer-rated Global Assessment of Functioning (GAF). When infant genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozygous G genotype, maternal depression diagnosis was significantly more likely to be observed in mothers of GG infants (X2(3)=8 .440,/)=.038). Mothers of GG infants had significantly lower GAF ratings than those with at least one A allele (r=.335, //=58, /;=.010), Infants displayed more optimally on the Child Responsiveness subscale of Responsiveness EAS scale when mothers were carriers of an A allele (F(l,59) = 3.984, p = .051). Furthermore, on the Affect subscale of the Sensitivity scale, mothers in concordant dyads (i.e. mother/infant GG/GG, AG/AG AA/AA) displayed significantly higher Affect after minority status, and mothers and infants genotypes had been separately taken into account, F(l,54)=5.685, p=.021, r|2=.095). An effect of genotypic concordance was also obtained on the Interaction subscale of the Sensitivity scale, after controlling for minority status and mothers and infants genotypes (F(l,54)=5.233, p=.026, r|2=.088). Similar findings were observed based on genotypic concordance on Non-Intrusiveness subscales of Following Child Lead (F(l,54)=4.246, p=.044, r|2=.072) and Adult Talking (F(l,54)=5.767, p=020, r|2=095).
Results of this study suggest that maternal genotype alone may not be enough to discriminate predictors of complex dyadic interactions. Biobehavioral synchrony between mother and infant, and specifically OXTR genotype concordance/discordance, may offer increased insight into the role oxytocin and OXTR play in observable dyadic emotional
v


availability, as well as the greater development of pair bonding, attachment, and social affiliation. Contrary to expectations from the literature, mothers with the GG OXTR genotype were not more sensitive or more prone to depression. If anything, this genotype was related to greater maternal intrusiveness during a semi-structured play interaction. However, GG mothers showed better sensitivity if their infants were GG than AG/AA, suggesting either that concordance promotes optimal biobehavioral synchrony and functioning of the oxytocin system or is a proxy for some other, unmeasured, variable that leads to better dyadic interactions. Possible mechanisms to explain the association between infant OXTR genotype and maternal depression, and between OXTR concordance and maternal emotional availability, are discussed.
The form and content of this abstract are approved. I recommend its publication.
Approved: Kevin Everhart
vi


DEDICATION
I dedicate this work to my wife, my son, and my parents. Jess: you are my biggest supporter and best friend. Your love, strength, and support have given me the motivation to continue to pursue my career goals while building a beautiful life together. Thank you for your unconditional love and understanding over the years. My graduate school experience has been so much more fulfilling because I have gotten to share it with you. Alfie: I love you more than youll ever know. You inspire me to be the best version of myself each and every day. Thank you for teaching me what its all about. Mom and Dad: I have spent a lot of time and energy throughout my academic career trying to gain a better understanding of infant and childhood development. I still have a lot to learn, but I am certain that none of the achievements throughout my life would have been possible without your love, support, and prayers. Thank you for all that you have given me, especially the confidence to believe in myself and never stop pursuing my dreams.
Vll


ACKNOWLEDGMENTS
I would like to thank the Infant Lab at the University of Colorado Denver, especially my mentors, Drs. Kevin Everhart and Peter Kaplan. I have benefitted from having two primary advisors that are each able to nurture my intellectual growth while offering constructive criticism and praise. Within the Infant Lab I would like to acknowledge the support of my fellow graduate student, Jo Vogeli. I appreciate all the time we have spent together pursuing shared interests and ambitions.
I would also like to thank Josh Fowler, Shiva Fekri, and Dr. Christopher Phiel. I am grateful for each of their contributions, which allowed for the successful processing of oxytocin receptor genotypes. Josh Fowler, in particular, deserves recognition for his assistance, guidance, and friendship while spending long hours in the lab together processing the genotypes that became a central focus of this dissertation.
Last, but not least, I would like to thank all the original members of my PhD cohort, Kellie Martins, Megan Grigsby, Stephanie Hooker, Carissa Kinman, and Jenn Altman. Each has been an inspiration to me over the years, and I am truly thankful to be able to call them my friends. In particular, I would like to thank Kellie, Megan, and Stephanie for making my time within the program one that has been filled with as much laughter as hard work.
vm


TABLE OF CONTENTS
CHAPTER
I. BACKGROUND.....................................................1
II. METHODS.......................................................17
Sample........................................................17
Participants..................................................18
Procedure.....................................................21
Measures......................................................24
Data Analysis Plan & Power....................................31
Ethical Considerations........................................32
III. RESULTS.......................................................34
Hypothesis 1: Genotype x Depression...........................34
Hypothesis 2: Genotype x EAS..................................36
Hypothesis 3: Genotype x Infant Performance...................37
Hypothesis 4: Concordance x EAS...............................40
IV. DISCUSSION....................................................43
Null Hypothesis Results.......................................44
Post Hoc Analysis.............................................46
Empirical Significance........................................49
Study Limitations.............................................51
Conclusion....................................................52
REFERENCES....................................................54
IX


APPENDIX
A. Concordance Poster Presentation...............................................67
B. Isohelix Swab Brochure........................................................68
C. Instructions for Isohelix DNA Isolation Kits..................................71
D. EAS Domain and Subscale Description...........................................73
E. COMIRB Certificate of Approval................................................76
x


LIST OF TABLES
Table
1. Demographic data by genotype........................................................20
2. Emotional Availability Scales and Subscale Descriptions............................29
3. Depression results by mother and infant genotype...................................35
4. EAS correlations of direct scores..................................................36
5. Non-Intrusiveness direct score MANCOVA result......................................37
xi


LIST OF FIGURES
Figure
1. Genetic variants in the oxytocin receptor gene (OXTR)...................................5
2. Biobehavioral synchrony in human attachments.............................................10
3. Power based on sample size for a 3x3 ANOVA...............................................32
4. BSID-III percentile rank by genotype....................................................39
5. Concordance by significant EAS subscale.................................................42


LIST OF ABBREVIATIONS
OXT
OXTR
CNS
PNS
PVN
SON
BDI-II
EPDS
PDSS
SCID
BSID-III
EAS
LS
PK
Cog
RC
EC
SE
COMIRB
Oxytocin
Oxytocin Receptor Central Nervous System Peripheral Nervous System Paraventricular Nuclei Suraoptic Nuclei
Beck Depression Inventory, 2nd Edition Edinburgh Postnatal Depression Scale Postpartum Depression Screening Scale Structured Clinical Interview for DSM-IV-TR Bayley Scales of Infant Development, 3rd Edition Emotional Availability Scales Lysis
Proteinase K Cognitive Scale
Receptive Communication Scale
Expressive Communication Scale
Social Emotional Scale
Colorado Multiple Institutional Review Board


CHAPTER I
BACKGROUND
The evolutionary roots of oxytocin (OXT) date back over seven hundred million years, as its important role in reproduction has existed, with few modifications, for both invertebrates and vertebrates (Donaldson & Young, 2008; Gimpl & Fahrenholz, 2001). In fact, the word oxytocin was derived from the Greek word okytokine, meaning quick birth, coined by the British pharmacologist Sir Henry Hallet Dale in 1906 after he discovered OXTs role in stimulating uterine contractions (Dale, 1906). Soon after, two separate laboratories reported OXTs role in lactation (Ott & Scott, 1910; Schafer & Mackenzie,
1911). Several decades later, laboratory studies performed by Vincent du Vigeaud at Cornell University allowed OXT to become the first human polypeptide hormone sequenced and biochemically synthesized (Vigneaud, Ressler, Swan, et al., 1953; Vigneaud, Ressler, Swan, Roberts, & Katsoyannis, 1954; Vigneaud, Ressler, & Trippett, 1953).
Due to this work, OXT is now understood to be a nonapeptide, or a single linear chain consisting of nine amino acids (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2) with a sulfur bridge between the two cysteines amino acids (National Center for Biotechnology Information, 2012a). Largely due to research involving animals models, and particularly rodents (Ludwig & Leng, 2006), OXT has been found to serve dual roles as both a neurotransmitter/neuromodulator in the central nervous system (CNS) and a hormone in the peripheral nervous system (PNS; Macdonald & Macdonald, 2010). Specialized cells in the paraventricular (PVN) and suraoptic (SON) nuclei of the hypothalamus produce OXT (Macdonald & Macdonald, 2010), which is then stored in the axon terminals of the posterior pituitary gland (Ross et al., 2009). Within the CNS, OXT exerts its central effects through
1


axonal connections from the parvocellular neurons in the PVN to critical brain regions, and through volume effects in areas with oxytocin receptors (Macdonald & Macdonald, 2010). In this way, oxytocins effects rely, at least in part, on both dynamic (i.e. hormone-driven) and static (i.e. genetically determined) changes to the location and density of receptors within the brain (Macdonald & Macdonald, 2010). Among the areas of the brain targeted by the release of OXT is the amygdala, which has a high concentration of OT receptors and whose functioning is associated with OXT levels, (Gimpl & Fahrenhholz, 2001; Macdonald & Macdonald, 2010). The release of the hormone and neurotransmitter OXT throughout the body and brain (Gimpl & Fahrenholz, 2001; Inoue et al., 1994) is believed to influence a wide-range of socioemotional functioning and social behavior (Campbell, 2010; Heinrichs, Von Dawans, & Domes, 2009; Ishak, Kahloon, & Fakhry, 2011; Kemp & Guastella, 2011; Uvanas-Moberg, Arn, & Magnusson, 2005).
OXTs role in socioemiotonal functioning and social behavior has been studied, and continues to be studied, extensively in animal models, and particularly in rodents (Insel, 2010). In female rats, maternal behavior has been shown to be regulated, at least in part, by OXT, and affiliative behavior in prairie voles has also been found to be mediated by the nonapeptide (Gimpl & Fahrenholz, 2001; Insel, 2010). Additionally, OXT has been associated with increased prosocial caregiving behaviors in humans. Given the importance of peripheral OXT production for childbirth and lactation, it is not surprising that prosocial parenting behaviors, especially those related to infant-attachment and pair-bonding, seem to correlate with OXT levels. From an evolutionary perspective, OXT plays a vital function in the production of offspring and maintenance of the offsprings health. Several studies have successfully demonstrated that the prosocial behavior of caregivers, especially practices of
2


breast-feeding, skin-to-skin contact (tender touch), and use of infant directed speech, are linked to circulating OXT levels (Barrett & Fleming, 2011; Feldman, Gordon, & Zagoory-Sharon, 2010; Feldman, Weller, Zagoory-Sharon, & Levine, 2007; Nelson & Panksepp,
1998; Swain, Lorberbaum, Kose, & Strathearn, 2007). Feldman et al., (2010) demonstrated that tender touch between mothers and infants, compared to only minimal contact, naturally increased salivary OXT levels between pre- and post- interaction saliva collections. Feldman et al., reported a similar finding for fathers, in respect to their own OXT levels, after shortterm stimulatory contact, compared to fathers that only engaged in minimal touch with their infants. Moreover, no significant difference was found between the maternal or paternal OXT levels observed (Feldman et al., 2010).
Although OXT has been found to have important implications for both animals and humans behaviorally, OXT is currently only known to have one receptor (OXTR) that is present in both the CNS and PNS (Gimpl & Fahrenholz, 2001; Lee, Macbeth, Pagani, & Young, 2009). The OXTR gene, localized on chromosome 3p253.3, has been found to encode the oxytocin receptor (National Center for Biotechnology Information, 2012b; Simmons, Clancy, Quan, & Knoll, 1995), which is known to be a G-protein coupled receptor that binds to the hormone/neuropeptide OXT and triggers a structural change to the receptor promoting the activation of downstream G proteins and the subsequent release of calcium ions from intercellular stores (Gimpl & Fahrenholz, 2001; Zingg & Laporte, 2003). Since OXT released from the pituitary gland is not able to re-enter the central nervous system due to the blood-brain barrier, it is believed that the behavioral effects of OXT are produced by the centrally projecting oxytocin receptors and expressed by neurons located throughout the brain and spinal cord (Ross et al., 2009). However, the specific effects of OXTRs activation
3


appear to vary by tissue, organ, and species (Zingg & Laporte, 2003), and the specific distribution of OXTR in the brain has been difficult to model as positron emission tomography radioligand have not yet been developed with appropriate CNS penetration or receptor specificity to accurately model, although some work in animal models continue to make progress in this area of research (Baribeau & Anagnostou, 2015). Furthermore, the study of OXTR is complex, as it has been found to have at least 28 unique single nucleotide polymorphisms (SNPs) across four exons and three introns (National Center for Biotechnology Information, 2012a).
Recent research has focused on two particular SNPs (i.e. rs53576 and rs2254298) in the third intron (Figure 1) that have been correlated with social affiliation, parenting, empathy, response to stress, and psychopathology (Bakermans-Kranenburg & van Ijzendoorn, 2014). In particular, SNP rs53576 has received growing attention for its connection to prosocial parenting behaviors (Bakermans-Kranenburg & Van Ijzendoorn, 2008; Klahr, Klump, & Burt, 2015), while rs2254298 is often studied in relation to symptoms of autism spectrum disorder (Francis et al., 2016). With this in mind, OXTR SNP rs53576 was selected for study in relation to mother-infant dyads.
4


Figure 1. Genetic variants in the oxytocin receptor gene (OXTR)
Oxytocin receptor
.Chr 3p25.3 n4fu ili
rmr
iniii
ii
5- -
T
ATG
rs237897 rs13316193 rs237889 rs2254298 rs2268494
*
TGA
rs53576
Untranslated region
' rs 1042778
Coding region Intron
Note: The (OXTR) gene is located on chromosome 3p25-3p26.2, spans 17 kb, and contains three introns and four exons. This 389 amino acid polypeptide with seven transmembrane domains belongs to the class I G protein-coupled receptor. Two single nucleotide polymorphisms (SNPs) in the third intron of OXTR have emerged as particularly promising candidates in the study of sociobehavioral phenotypes (indicated in bold): rs53576 (G/A) and rs2254298 (G/A). The main SNPs with their location and rs number are shown above, and rs53576, the focus of this study, is denoted with a star. Exons are indicated by the darker shaded boxes, and the untranslated regions are shown by the lighter boxes. Variants in the gene are shown by arrows. Chr, chromosome (Kumsta & Heinrichs, 2013).
Genotypic polymorphism of OXTR SNP rs53576 is expressed through the homozygous or heterozygous pairing of alleles A and G (Rodrigues, Saslow, Garcia, John, & Keltner, 2009). Although the underlying processes linking variants of the OXTR gene to actual OXT levels in humans have not yet been clarified, at least one study has suggested that OXTR rs53576 may contribute to oxytocins ability to bind with its receptor (Tost et al., 2010). Despite the uncertainty regarding the underlying mechanism that may relate OXTR to socioemotional functioning and social behavior, comparison of OXTR genotypes have suggested that carriers of an A allele (i.e. G/A and A/A) display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, relatively poor parenting skills, decreased empathy and deleterious mental health outcomes compared to individuals
5


with a homozygous G/G allele pair (Bakermans-Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010). Interestingly though, depressive symptomatology may suppress the positive effects of the homozygous G/G allele pair (Riem et al., 2011). Despite the reported benefits of parenting behaviors associated with the homozygous G/G OXTR allele pair, it has also been associated with a greater risk of developing major depression (R. J. Thompson et al., 2011). A 2013 study (N=288) reported that carriers of a G allele, particularly in the presence of early life adversity, were associated with increased vulnerability to depression symptoms (McQuaid, Mclnnis, Stead, Matheson,
& Anisman, 2013), which contrasted the results of an early study (2011) that linked depression symptomatology to carriers of an A allele (Saphire-bernstein, Way, Kim,
Sherman, & Taylor, 2011). However, a longitudinal study (N=155) conducted by Feldman et al. found that not only did depressed mothers show decreased levels of OXT, but chronically depressed mother-child dyads were four times more likely to have a homozygous G/G OXTR allele pair than non-depressed mothers (Feldman, 2012b).
Although individual studies have supported the idea of OXTR being associated with sociability, parenting behaviors, and psychopathology, it remains unclear how generalizable these findings are despite their reported statistical significance. Like many genetic association studies, results can often be difficult to replicate (Ioannidis, Ntzani, Trikalinos, & Contopoulos-Ioannidis, 2001). As the published literature regarding OXTR rs53576 has grown, meta-analytic studies have been completed on OXTRs purported role in sociability and psychopathology. A 2014 meta-analysis included the reported effect sizes for 52 samples (N = 17,557 participants) that analyzed OXTR rs53576 in relation to one or more of five
6


distinct domains (i.e. biology, personality, social behavior, psychopathology, and autism) as categorized by the authors (Bakermans-Kranenburg & van Ijzendoorn, 2014). Based on their systematic review of the OXTR rs53756 literature and subsequent meta-analysis, none of the five domains demonstrated a significant main effect with OXTR, which led the studys authors to conclude that OXTR SNP rs53576 does not significantly explain the complexities of human social behavior.
However, an even more recent systematic review and corresponding meta-analysis challenged these results, particularly as OXTR rs53576 relates to sociability (Li et al., 2015). Li et al. (2015) deconstructed the social behavior domain used in Bakermans-Kranenburg & van Ijzendoorns (2014) study and recategorized phenotypic outcomes reported in all relevant published research into a general sociality domain (e.g. extraversion, empathy, and social loneliness), or a sociality within the context of close relationships domain (e.g. maternal sensitivity, child/adult attachment, and marital quality), as well as teasing apart reported effects on depression from a larger psychopathology domain. Utilizing data available from eighteen studies and twenty-four unique samples (N=4,995) that reported phenotypic outcomes related to general sociality, the authors found that homozygous GG allele OXTR genotypes demonstrated increased social attributes compared to carriers of an A allele. However, a significant result was not found when a separate analysis of ten studies and fifteen independent samples (N=5,262) was conducted to look at the association between OXTR rs53576 and quality of close relationships. Collectively, these meta-analytic results led Li et al. (2015) to posit that OXTR rs53576 polymorphism predicts how an individual will generally relate to others, but not within the unique context of close relationships.
7


Moreover, both Bakermans-Kranenburg & van Ijzendoorn (2014) and Li et al.s (2015) meta-analyses failed to show an association between OXTR and psychopathology, specifically, depression. However, it should be noted that the samples included within their respective meta-analyses were quite heterogeneous, including samples from different countries, both sexes, and diverse psychiatric populations. Biological and epidemiological evidence suggests that PPD is unique to other forms of depression with regard to biopsychosocial risk factors (Kimmel et al., 2016), and previous research has demonstrated that OXTRs genotype is expressed phenotypically differently dependent on cultural norms (Feldman, Monakhov, Pratt, & Ebstein, 2016; H S Kim et al., 2011). No study has specifically reported on the association between OXTR rs53576 and PPD status, although a recent study demonstrated an interaction between perinatal OXTR methylation and development of PPD (Bell et al., 2015; Serati, Redaelli, Buoli, & Altamura, 2016).
Therefore, it is worth continuing to investigate associations between OXTR and PPD, despite the null findings of recent meta-analytic studies regarding OXTR and depression phenotype.
Although meta-analytic studies have drawn concern for OXTRs purported role in sociability and psychopathology, and even the quality of close relationships, published studies continue to report associations between OXTR rs53576 and parenting (Bakermans-Kranenburg & Van Ijzendoorn, 2008; Klahr et al., 2015; Marsh et al., 2012; Michalska et al., 2014). In Li et al.s (2015) meta-analysis, which did not find a significant association between OXTR and sociality within the context of close relationships, we again see a heterogeneous mixture of diverse adult focused samples included in their analysis of OXTR in relation to close relationships. Phenotypic outcome measures of the included samples ranged from pair-bonding (Walum et al., 2012), separation anxiety (Costa et al., 2009), and
8


attachment security/anxiety (Chen & Johnson, 2012; Costa et al., 2009; Gillath, Shaver,
Baek, & Chun, 2008; Krueger et al., 2012; Luijk et al., 2011; Rodrigues et al., 2009) to marital discord (Bakermans-Kranenburg & Van Ijzendoorn, 2008), interpartner conflict (Sturge-Apple, Cicchetti, Davies, & Suor, 2012), and romantic relationship security (Raby, Cicchetti, Carlson, Egeland, & Collins, 2013). Although both recent meta-analyses included reported effect sizes from a multitude of published studies that demonstrated phenotypic outcomes related to parent-child relationships, parenting behavior was not the specific focus when considering sociability or even sociability within the context of close relationships. Perhaps just as Li et al. (2015) proposed an inherent difference between general sociability and sociability within the context of a close relationship, there may be a significant difference in the functioning of the oxytocinergic system between mother-infant dyads.
Recent work has led to an appreciation for a complex synchronous relationship that occurs between an individuals environment and biological factors that can influence both the mother and infant in the perinatal and postpartum sensitive periods, particularly in relation to bond formations, parenting behaviors, and attachment (Feldman, 2016). Insight from early rodent and animal models into the association between pair-bonding and the oxytocin system has continued with the study of humans (Carter, 2014). A leading conceptualization of the role oxytocin plays on human attachment formation stresses the importance of biobehavioral synchrony, or a coordinated biological and behavioral process that occurs during social interactions between mother-infant dyads (Figure 2; Feldman, 2017). This conceptualization is noteworthy within the study of mother-infant dyads, as the oxytocinergic system is being shaped in infancy. In fact, reports have demonstrated that behavioral interactions between mother-infant dyads in early life are partially responsible for the
9


organization of oxytocin availability and OXTR localization in the brain (Dumas, Nadel, Soussignan, Martinerie, & Gamero, 2010; Weaver et al., 2004). From an epigenetic framework, this early life experience and the corresponding oxytocinergic system development shapes the ability for an individual to go on to form attachments and ultimately parent the next generation (for a review of the neurobiology of human attachments, please see Feldman, 2017).
Figure 2. Biobehavioral synchrony in human attachments
Biobehavioral synchrony in human attachments
Parents
Behavioral synchrony Heart rate coupling Endocrine fit Brain-to-brain synchrony
Synchronized Synchronized HR Coordinated OT Coordinated brain
behavior in gaze, during synchronized response following oscillations in alpha
affect, vocal, and interactions contact and gamma rythms
touch
Mother-specific Coordinated cortisol
father-specific response to stress
Note: Human attachments are characterized by the coupling of the partners physiological and behavioral processes during moments of social contact. Such coupling is observed across four systems: matching of nonverbal behavior, coupling of heart rhythms and autonomic functioning, coordination of hormonal release, and brain-to-brain synchrony. Abbreviations: HR, heart rate; OT, oxytocin (Feldman, 2017).
Several previously published studies have specifically reported on associations between OXTR rs53576 genotype and maternal behavior. In a pioneering study published in 2008, Bakermans-Kranenburg & van Ijzendoorn became the first to report a relationship between mothers (N=159) carrying a homozygous G allele genotype and increased sensitive parenting. A study then came out linking non-mothers with low depression scores (N=40) to increased heart rate responses to infant cries when carriers had the GG genotype (Riem et al.,
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2011). Marsh et al. (2012) followed with a significant finding associating mothers (N=57) with increased infant face preference following intranasal OXT administration in carriers of the homozygous G allele sequence. A somewhat more complex study (N=40) followed that retrospectively associated maternal carriers of the A allele with increased levels of prosocial parenting (observed when the child was 4-6 years of age) and neural activation of prosocial parenting regions of the brain (i.e. bilateral orbitofrontal cortex and left anterior cingulate cortex) when viewing photographs of their children fifteen years later (Michalska et al., 2014). Although Michalska et al.s (2014) finding contradicts prior studies demonstrating a link between the heterozygous G OXTR rs53576 genotype and prosocial parenting behaviors, it did still report a significant finding between OXTR polymorphism and parenting. Perhaps most recently, Klahr et al. (2015) reported that in a sample of five-hundred twin families (N=1000 twins, n=494 mothers), maternal carriers of the homozygous G allele predicted greater warmth with her offspring, even after controlling for the childs genotype. This latter study was novel, in that it considered both direct (i.e. maternal genotype) and indirect (i.e. child genotype) genetic influences (Klahr et al., 2015). The indirect genetic influences of the child were considered due to meta-analytic findings highlighting the importance of not only parental, but also child, genetic factors on parenting behaviors due to an evocative gene-environment correlation (Klahr & Burt, 2014, p.547). Although these findings are quite notable and speak to the potential importance of considering dyadic gene x environment research designs, Klahr et al. (2015) utilized a complex measurement system involving confirmatory factor analysis based on two objective parent/child coding systems (i.e. joystick coding method and the more global Parent Child Interaction System coding method) for obtaining parent composite scores statistically
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associated with warmth, control, and negativity dimensions that mapped onto prior behavioral genetic studies of parenting originating from their workgroup (Klahr & Burt, 2014).
However, few studies have specifically addressed the relationship between genotypic and phenotypic expressions of OXTR rs53576 on maternal depression and in relation to dyadic emotional availability. The construct ofemotional availability was originally used to describe the unique interaction between a mother and child, while emphasizing the role of emotional expression on dyadic interactions (Emde, 2012). Emotional availabilitys early conceptualization incorporated emotional expression and responsivity into work regarding attachment theory (Bowlby, 1953, 1969, 1973, 1979, 1980). Since then the construct of emotional availability has been reconceptualized based on research regarding the quality of parent-child interactions (Biringen, 2000), and is now used in the research setting to describe dyadic interactions across several parental dimensions (Sensitivity, Structuring, Non-Intrusiveness, Non-Hostility) and two child dimensions (Responsiveness to parental bids for attention and Involvement of the parent in play). Given the above overview of empirical support for a potential relationship between OXT and OXTR on prosocial parenting behaviors and sensitivity, it is believed that OXT and OXTR may influence observed emotional availability. For instance, a recent study conducted in the Netherlands demonstrated a causal linkage between intranasal OXT administration and paternal emotional availability (Naber, van Ijzendoom, Deschamps, van Engeland, & Bakermans-Kranenburg, 2010), and, most notably, Bakermans-Kranenburg & van Ijzendoorns (2008) pioneering study looking at prosocial parenting behaviors demonstrated that mothers homozygous for the G allele pair of OXTR SNP rs53576 exhibited increased sensitivity.
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The focus of this study was to examine the synchronous relationship that may exist between mothers, with or without depression, and their infants. The polymorphic arrangement of OXTR rs53576 allele pairings may be associated with an increased risk of poor infant development if confronted with environmental adversity (maternal depression), as supported by prior studies. Meaney (2001) illustrated a theoretical model for the cross-generational transmission of OXTR genotypic expression related to impoverished maternal care. Their model was largely based on empirical reports investigating the effects of environmental stress on maternal care in bonnet macaque populations and rodents (Coplan et al., 1996, 1998; Meaney, 2001; Rosenblum & Andrews, 1994), however more recent findings in human mother-infant dyads suffering from postpartum depression may offer additional support for Meaneys model. In one study, roughly 60% of the children raised by depressed mothers would develop a psychiatric disorder within the first six years of life (Feldman, 2012b), but children of depressed mothers that carried at least one copy of an A OXTR allele had increased OXT levels, significantly reduced psychopathology, and expressed empathy that was comparable to a control population (Feldman, 2012b). This finding, in part, led Feldman to posit that in the context of risk associated with maternal-infant bonding, more optimal functioning of the [oxytocin] system may promote resilience and provide a buffer that mitigates some of the effects of maternal depression on childrens social competence, empathy, and proneness to psychopathology (Feldman, 2012b, p. 387). However, this finding is contrasted by a study that reported that children (N=441) inheriting an A OXTR allele, while raised under the presence of maternal depression (n=41), are more susceptible to developing depression themselves in their adolescence (Thompson, Hammen, Starr, & Najman, 2014). By analyzing maternal depression in relation to the emotional availability of
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mother-child dyads we can attempt to distinguish how OXTR polymorphic variations on SNP rs53576 influences dyadic emotional availability development under adverse environmental conditions (postpartum depression), which may serve as an important antecedent condition for the contrasting reports on the association between OXTR and the intergenerational transmission of psychopathology.
Hypothesis and specific aims
This study was designed to determine the genotype of OXTR rs53576 for a population of mother-child dyads. The studys aim was to correlate specific OXTR genotypes with maternal depression in the postpartum period, dyadic emotional availability ratings, and infant development. Within this objective, four specific hypotheses were investigated:
1. Genotype x Depression. Despite the finding of the more recent meta-analysis suggesting that there is not an association between OXTR rs53576 and depression (Li et al., 2015), this study when originally designed predicted that the presence of depression in the postpartum period will be related to the presence of the G/G OXTR allele sequence in the mother, similar to Thompson et al.s (2011) and McQuaid et al.s (2013) findings linking depression symptomatology with OXTR genotype.
2. Genotype x EAS. Given the published literature suggesting that carriers of an OXTR rs53576 A allele (i.e. A/G and A/A) display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, relatively poor parenting skills, and decreased empathy compared to individuals with a homozygous G/G allele pair (Bakermans-Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010), this study predicted that mothers
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carrying an A OXTR genotype will demonstrate decreased levels of Sensitivity as measured by the Emotional Availability Scales.
3. Genotype x Infant Performance. Recent literature studying mother-child dyads in relation to OXTR have suggested that there may be a protective factor associated with the genotype of the child (Feldman et al., 2010; S. M. Thompson et al., 2014). Although published findings appear to contrast as to whether an A or a G allele predicts more optimal socioemotional functioning of the child, this study attempted to operationalize the protective factor outcome by analyzing infant genotype in relation to two performance based assessments (i.e. Child Responsiveness direct score on the EAS and Cognitive Development score on the Bayleys Scales of Infant Development) collected as part of this study. As such, this study predicted that infants of depressed mothers who are carriers of an A OXTR allele inherit a protective genetic factor leading to:
a. increased ratings of Child Responsiveness on the Emotional Availability Scales.
b increased Cognitive Development scores, as measured by the Bayley s Scales of Infant Development, 3rd Edition, relative to those without an A allele.
4. Concordance x EAS. A conceptualization of the oxytocinergic system in dyads relying on biobehavioral synchrony for optimal functioning (Feldman, 2012a), coupled with literature suggesting that homozygous G carriers demonstrate more optimal prosocial parenting behaviors (Bakermans-Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al.,
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2010), led to the original hypothesis that non-depressedmother-chdd dyads that are both carriers of the homozygous G genotype will demonstrate significantly higher dyadic emotional availability ratings than dyads that did not meet this criterion.
Interestingly, though, since these original hypotheses were made, Klahr et al. (2014,
2015) has offered compelling evidence that supports a conceptual biobehavioral synchrony model (Feldman, 2012a) for considering evocative gene x environment interactions in gene association studies. Moreover, studies associating child OXTR genotype to positive outcomes (Feldman et al., 2010; S. M. Thompson et al., 2014) makes one inquire about the bidirectional effect that child genotype might have on parental socioemotional functioning, or vice versa. At around the same time Klahr et al. and Thompson et al. published their studies, our own lab utilized pilot data from this study in preparation of a poster presentation submission (Appendix A) that showed emerging evidence for an effect on the concordance, or sameness, between mother-child OXTR genotypes with maternal depression and dyadic emotional availability (Asherin et al., 2016). To further investigate the possible role that genotype synchrony may have on assessments of maternal depression and dyadic emotional availability, posthoc analysis was conducted that considered the infant genotype in relation to maternal depression, maternal and infant genotype effects on dyadic emotional availability separately, as well as categorically defined based on dyadic genotype concordance (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant). Posthoc analysis results are included within the hypothesis that was being tested that directly related to the measures of depression, emotional availability, and infant development.
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CHAPTER II
METHODS
Sample
A prospective observational case-control design was utilized for this study to draw inferences about an antecedent condition (OXTR genotype) that may be associated with levels of a dependent variable (maternal psychopathology, dyadic emotional availability, and infant development) (Kazdin, 2003). Cases were considered participants with a current or past major depressive episode, as this diagnostic information was collected along with the additional study relevant dependent variables as part of a larger study aimed to assess infants attention to infant directed speech and cognitive development based on the depressive status of caregivers (Kaplan et al., 2014; Kaplan, Danko, Diaz, & Kalinka, 2011; Kaplan, Danko, Kalinka, & Cejka, 2012; Kaplan, Sliter, & Burgess, 2007). Data providing the basis for classifying participants with respect to the antecedent condition (OXTR genotype) were collected after participants signed an additional consent form for the collection of salivary samples from both mothers and infants. All research related methods and procedures were approved by the local university affiliated institutional review board (COMIRB protocol #11-1641 & COMIRB protocol #14-0851). Mothers with and without a history of depression were included in the study. Inclusion criteria was infants between 3-24 months, with mothers that are 18 years of age or older. Participants were screened and excluded, prior to enrollment, if the infant had a history of serious medical complications, but no participants had to be excluded on this criterion. A total of sixty-two mother-infant dyads consented to have their salivary samples collected, but one dyad was not able to provide a sample due to study personnel error in not collecting the sample prior to them exiting the study. Of the
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remaining sixty-one dyads, genotypic information was collected and OXTR genotype was identified for all but one infant. Once in the study protocol, the mother and infant dictate the studys progression breaks, diaper changes, feedings, fussiness, or need for naps caused some participants to exit the study prior to all data being collected. However, dependent variables were collected for the majority of dyads with all dyads (N=61) providing demographic information, 60/61 mothers completing a depression screener, 59/61 mothers undergoing a structured clinical interview, 60/61 completing the dyadic emotional availability task, and 57/61 infants completing the measure of cognitive development. All available data for the sixty-one mother-infant dyads was included in the resulting analyses.
Participants
Table 1 presents demographic information for the full sample of mothers and infants (N = 61) that had salivary samples collected as part of this study. All participating mothers (n(mom)=61) had OXTR rs53576 successfully genotyped by utilizing the salivary collection, DNA isolation, and genotyping procedure detailed below. One infants genotype was considered unidentifiable after two attempts at genotyping, and, thus, subsequently excluded from final analyses (n(infant)=60).
No significant differences were found as a function of maternal genotype (i.e. GG, GA, or AA) on infant gender, maternal age, infant age, ethnicity, marital status, maternal level of education, income, number of offspring, depression screening score, depression diagnosis, or Global Assessment of Functioning (GAF) scores as recorded during the structured clinical interview. When carriers of an A allele (i.e. GA/AA) were collapsed into a distinct group, minority status was found to be significantly higher compared to those with a homozygous G allele pair (t(58)=-2.544; p=0.014).
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No significant differences were found as a function of infant genotype (i.e. GG, GA, or AA) on infant gender, maternal age, infant age, ethnicity, marital status, income, number of offspring, or depression diagnosis. When carriers of an A allele (i.e. GA/AA) were collapsed into a distinct group, maternal education level was found to be significantly higher compared to those with a homozygous G allele pair (t(58)=-2.332; p=0.023). Additionally, infant genotype was found to be significant in relation to measures of depression and GAF as discussed under the results for Hypothesis 1: Genotype x Depression.
No significant differences were found as a function of dyadic genotype concordance on infant gender, maternal age, infant age, ethnicity, marital status, income, number of offspring, depression screening score, depression diagnosis, or GAF scores. However, maternal education level was found to be significantly higher in discordant dyads compared to those categorized as concordant (t(58)=-2.271; p=0.027).
Additionally, the null hypothesis of Hardy-Weinberg equilibrium cannot be rejected for either maternal (x2=1.53; p=0.22) or infant (x2=0.12; p=0.73) genotype (Rodriguez, Gaunt, & Day, 2009), suggesting that the study sample did not significantly deviate from an expected genetic equilibrium inherent in the population and that results were unlikely to be due to errors in genotyping (Hosking et al., 2004).
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Table 1. Demographic data by genotype
Variable Maternal Genotype Infant Genotype Concordant
N = 61 dyads GG GA AA GG GA/AA GG GA AA GG GA/AA Yes No
fr(mom) 61 33 21 7 33 28 33 21 7 33 28 37 23
fr(infant) 60 31 25 4 31 29 31 25 4 31 29 37 23
Infant Gender (f/m) 16/15 12/13 2/2 16/15 14/15 16/15 12/13 2/2 16/15 14/15 21/16 9/14
Age of mother (years) 27.8 (4.6) 28.1 (5.0) 30.4(3.4) 27.8 (4.6) 28.71 (4.7) 27.8 (4.7) 28.7(4.8) 28.75 (1.7) 28.7(4.5) 27.8 (4.7) 28.1 (5.0) 28.6 (3.9)
Age of Infant (days) 211 (83.6) 241 (91.9) 270 (90.0) 211 (83.6) 245 (90.6) 211 (83.6) 241 (91.9) 270 (90.0) 211 (83.6) 245 (90.6) 219.8(90.3) 240.4 (84.^
Ethnicity
White 29 (87.9%) 13(61.9%) 4 (57.1%) 29 (87.9%) 17 (60.7%) 26 (83.9%) 18(72.0%) 2 (50.0%) 26 (83.9%) 20 (69.0%) 31 (83.8%) 15 (65.2%
Minority 4(12.1%) 8(38.1%) 3 (42.9%) 4(12.1%) 11 (39.3%)* 5 (16.1%) 7 (28.0%) 2 (50.0%) 5 (16.1%) 9(31.0%) 6 (16.2%) 8 (34.8%)
African American 0 (0.0%) 2 (9.5%) 1 (14.3%) 0 (0.0%) 3 (10.7%) 1 (3.2%) 1 (4.0%) 0 (0.0%) 1 (3.2%) 1 (3.4%) 0 (0/0%) 2 (8.7%)
Asian 1 (3.0%) 2 (9.5%) 0 (0.0%) 1 (3.0%) 2(7.1%) 1 (3.2%) 2 (8.0%) 0 (0.0%) 1 (3.2%) 2 (7.0%) 1 (2.7%) 2 (8.7%)
Latina 1 (3.0%) 4 (19.0%) 0 (0.0%) 1 (3.0%) 4 (14.3%) 2 (6.5%) 2 (8.0%) 1 (25.0%) 2 (6.5%) 3 (10.3%) 3 (8.1%) 2 (8.7%)
Native American 2(6.1%) 0 (0.0%) 2 (28.6%) 2(6.1%) 2(7.1%) 1 (3.2%) 2 (8.0%) 1 (25.0%) 1 (3.2%) 3 (10.3%) 2 (5.4%) 2 (8.7%)
Marital Status
Married 22 (66.7%) 12(57.1%) 6 (85.7%) 22 (66.7%) 18 (64.3%) 23 (74.2%) 13 (52.0%) 4 (100%) 23 (74.2%) 17 (58.6%) 23 (62.2%) 17 (74.0%
Unmarried 11 (33.3%) 8(38.1%) 1 (14.3%) 11 (33.3%) 9 (32.1%) 8(25.8%) 11 (44.0%) 0 (0.0%) 8(25.8%) 11 (38.0%) 13 (35.1%) 6 (26.0%)
Mothers Education 5.2 (1.2) 5.4 (1.1) 6.1 (0.7) 5.2 (1.2) 5.6 (1.1) 5.1 (1.0) 5.8 (1.1) 5.8 (1.3) 5.1 (1.1) 5.8 (1.1) 5.2 (1.2) 5.8 (0.9)*'
Family Income 6.4 (2.1) 6.2 (2.6) 6.7 (2.2) 6.4 (2.1) 6.4 (2.5) 6.1 (2.2) 6.8 (2.3) 6.8 (2.5) 6.1 (2.2) 6.8 (2.3) 6.1 (2.4) 7.0 (1.9)
Number of children 1.5 (0.6) 1.4 (0.5) 1.6 (0.8) 1.5 (0.6) 1.4 (0.6) 1.5 (0.6) 1.5 (0.7) 1.0 (0.0) 1.5 (0.6) 1.4 (0.6) 1.4 (0.5) 1.7 (0.6)
Depression Screen
BDI-II score 12.3 (9.8) 10.2(6.8) 8.4 (3.8) 12.3 (9.8) 9.7 (6.1) 13.2(10.2) 9.2 (5.6) 7.7 (3.1) 13.2(10.2) 9.0 (5.3) 12.5 (9.0) 9.2 (7.2)
PDSS score 63.1 (21.5) 68.3 (25.0) 51.9(13.3) 63.1 (21.5) 64.0 (22.2) 67.3 (26.5) 58.7(16.6) 65.3 (13.1) 67.3 (26.5) 59.6(16.1) 66.2(21.4) 59.7(23.8
EPDS score 6.8 (5.6) 6.1 (4.1) 3.7 (1.7) 6.8 (5.6) 5.5 (3.78) 7.7 (5.7) 4.8 (3.3) 4.3 (2.5) 7.7 (5.7)" 4.8 (3.2) 7.1 (5.1) 4.8 (0.9)
SCID Depression Dx
No Depression 10 (32.3%) 6 (30.0%) 3 (60.0%) 10 (32.3%) 9 (36.0%) 6 (19.4%) 13 (52.0%) 0 (0.0%) 6 (19.4%) 13 (44.8%) 10 (27.0%) 9(39.1%;
Full Remission 14 (45.2%) 12 (60.0%) 2 (40.0%) 14 (45.2%) 14 (56.0%) 15 (48.4%) 9 (36.0%) 3 (75.0%) 15 (48.4%) 12(41.4%) 15 (40.4%) 12 (52.2%
Partial Remission 3 (9.7%) 0 (0.0%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 3 (8.1%) 0 (0.0%)
Current Depression 4 (12.9%) 2 (10.0%) 0 (0.0%) 4 (12.9%) 2 (8.0%) 5 (16.1%) 1 (4.0%) 0 (0.0%) 5 (16.1%) 1 (3.4%) 5 (13.5%) 1 (4.3%)
GAF rating 74.2(11.4) 76.2(11.0) 78.8(11.9) 74.2(11.4) 76.8(11.1) 71.9(11.1) 80.5 (10.4)" 73.3 (7.9) 71.9(11.1) 79.4(10.3)" 74.2(11.3) 77.3 (11.3
Note: Columns are defined based on genotype. Last two columns of maternal and infant groups compare homozygous G carriers with carriers of a minor allele. Last two columns of table compare dyads with concordant genotypes with those dyads that are not concordant. Numbers in parentheses are standard deviations, unless denoted as a percentage of the sample. denotes p=.01 and ** denotes p<05. Abbreviations: Beck Depression Inventory, 2nd Edition (BDI-II), Postpartum Depression Screening Scale (PDSS), Edinburgh Postnatal Depression Scale (EPDS), Structured Clinical Interview for DSM-IV-TR (SCID), Diagnosis (Dx), GAF = Global Assessment of Functioning (GAF), as estimated from SCID interviews.
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Procedure
Participants enrolled in COMIRB protocol #11-1641 were invited to participate in the current study. If participants chose to take part in this study (COMIRB protocol #14-0851), both mother and child were consented to the current project, allowed a chance to ask any questions they may have about participating, and then asked to provide a salivary sample.
The salivary sample was necessary to determine an individuals OXTR genotype. OXTR genetic information was then analyzed in relation to measures completed while participating in COMIRB protocol #11-1641. The measures recorded in COMIRB protocol #11-1641 that are relevant to the aims of this study included demographic questionnaires, self-report measures of depression (i.e. Beck Depression Inventory, 2nd Edition (BDI-II), Postpartum Depression Screening Scale (PDSS), Edinburgh Postpartum Depression Scale (EPDS), and a Structured Clinical Interview for Diagnosis (SCID-IV), infant development as measured by the Bayley Scales of Infant Development, 3rd Edition (BSID-III), and dyadic emotional availability as measured by observational coding of an unstructured play session (Emotional Availability Scales; EAS). Participants were thanked for participating in this study, but they were not offered financial compensation above and beyond the payment they received for participating in COMIRB protocol #11-1641 (i.e., $50.00). No additional payment was offered for participating in this study in an effort to ensure that subjects did not feel coerced to provide their genetic information.
Saliva/Buccal Cell Collection: A cheek swab/saliva sample was taken from the participant (i.e. mother and child). The sample was collected after the subjects had abstained from eating or drinking for 45-60 minutes to ensure a clean sample was obtained, based on the
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recommendation of the manufacturer of the buccal cell collection kits, Isohelix. The collection kit (SK-1 Isohelix swab) used was essentially an oversized cotton swab (see Appendix B). After the sample was collected (approximately 60 seconds of swabbing per individual) the unlinked, numerical value given to subjects for participating in COMIRB protocol #11-1641 was written on the tube containing the saliva sample. The identity of the participant was protected since there was no link between the numerical value and the identity of the participant.
DNA Isolation: In order to genotype the participants for the OXTR gene (oxytocin receptor gene) rs53576, it was imperative to first isolate the DNA in the saliva sample (see Appendix C). Since the DNA is located inside the cells themselves, DNA was extracted out of the cells by adding a lysis buffer. A lysis (LS) buffer creates an environment in which the amount of water outside the cell is greater than the amount inside the cell, which causes water to move into the cells through a process called osmosis. Through this process, the cell eventually bursts open exposing DNA. Five-hundred microliters of the LS buffer was added to the tube containing the buccal swab (saliva sample) in order for the osmosis process to occur. Although the DNA was exposed at this point, there was still a lot of other biological material floating around, including proteins and nucleases (nucleases cleave the bonds holding DNA together, which are unwanted for continued DNA isolation). In order to remove this material, a solution called proteinase K, or PK solution for short, was added to the tube containing the buccal swab. The PK solution digests proteins and nucleases while leaving DNA intact by exploiting a fundamental difference between proteins and nucleases and DNA. Proteins and nucleases have amines, which contain nitrogen, on their molecular structure while DNA has
22


phosphates (called phosphodiester bonds). The PK solution is attracted to the nitrogen on the proteins and nucleases and dissolves them upon bonding. Twenty microliters of the PK solution were added and the tube was vortexed briefly in order to accomplish this separation. DNA isolation was continued, either on the same day or after storage (the buccal swab sample and collection tube, with the added LS buffer and PK solution, can be stored at room temperature for up to 3'A years before continuing with DNA isolation and genotyping procedures), by placing the tube in a 60C water bath for one hour (this step ensures the PK solution has had time to digest the unnecessary proteins and nucleases). The resultant solution is removed from the original tube and placed in a 1.5-milliliter centrifuge tube. To this solution a capture buffer, or CT solution, was added to the centrifuge tube containing the sample. The capture buffer controls the pH of the solution and enables the DNA to be separated from the rest of the material. After the 400 microliters of CT solution was added, the centrifuge tube was vortexed briefly and then placed in a microcentrifuge to spin at 13,000 rpm for 7 minutes. This process causes the heavier DNA molecules to go to the bottom of the centrifuge tube. The DNA that ends up on the bottom is called the pellet. The rest of the material/solution was removed by pipette leaving the isolated DNA pellet intact. Finally, a re-hydration buffer, or TE solution, was added to the isolated DNA. The TE solution helps stabilize the DNA and prevent degradation. After 150 microliters of this solution was added, the sample was ready for genotyping.
OXTR genotyping: Once the DNA was ready for genotyping, 2 microliters containing 10 nanograms of DNA was removed from the isolated sample and placed in a separate polymerase chain reaction, or PCR, tube. PCR is a biochemical technology in molecular
23


biology that is used to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In this case, the OXTR gene is sequenced through the use of a STEPONE qPCR machine. In order to prepare the DNA sample for qPCR, a master mix containing DNA primers, TAQ polymerase, probes, and water was added to the PCR tube containing the DNA sample. The tube was placed into the machine where it initially heated up the DNA. The heating process unzips the double helix DNA strand leaving single DNA strands in the solution, through a process called denaturation. The primers in the master mix isolate the portion of DNA containing the OXTR gene. In the case of the OXTR gene it is 348 of the 3 billion base pairs that make up DNA that will be isolated by the primers. TAQ polymerase then replicates these isolated portions of DNA amplifying the OXTR SNP rs53576. The binding of the probes in the master mix indicates which alleles are present in each sample. The alleles present determine the OXTR genotype for SNP rs53576 (i.e. G/G, G/A, or AJA) for each participant. With 2 probes present, one only binds to the G allele and the other only binds to the A allele. Located on the probes are molecules that fluoresce, or simply light up, when they bind to specific parts of DNA. In their unbounded state they do not fluoresce, as if there is a lens cover over a flashlight. Once they bind to the specified allele (i.e. G or A), however, they each emit a unique wavelength of light that is measured by the STEPONE qPCR machine. This is done to determine the genotypes of the participants for the OXTR gene SNP rs53576.
Measures
Self-report and structured clinical measures of depression, an assessment of dyadic emotional availability, and an assessment of infant development were collected, in addition
24


to DNA collection and subsequent OXTR genotyping, as described in the procedures section above. Details of each additional measure that was included in this study are provided below.
Assessment of Depression: Self-reported symptoms of depression was obtained using the Beck Depression Inventory, 2nd Edition (BDI-II; A. T. Beck, Steer, & Brown, 1996), Edinburgh Postnatal Depression Scale (EPDS; Cox, Holden, & Sagovsky, 1987), and the Postpartum Depression Screening Scale (PDSS; Beck & Gable, 2000). Additionally, a Structured Clinical Interview for DSM-IV-TR (SCID; First, Spitzer, Gibbon, & Williams, 2002) was used to assess current and past psychopathology.
BDI-II. The Beck Depression Inventory, 2nd Edition (BDI-II) is a proprietary self-report assessment developed as a revision to the original Beck Depression Inventory (BDI) published by Psychological Corporation (A. T. Beck et al., 1996). The BDI-II, like its predecessor the BDI, is a twenty-one item self-report measure developed to detect symptoms of depression. The BDI-II revision made changes reflecting revised diagnostic criteria included in the DSM-IV for major depressive disorder, i.e. changes made to item location, wording, and time frame (Carvalho Bos et al., 2009; Dozois, Dobson, & Ahnberg, 1998; Ward, 2006). Empirical literature consistently demonstrates that the BDI-II is an accurate measure of depression, occurring in the postpartum period or otherwise (Boyd, Le, & Somberg, 2005; Chaudron et al., 2010; Dozois et al., 1998; Smarr & Keefer, 2011). A review of self-report instruments used for PPD found the BDI-II to have excellent internal consistency, excellent specificity, acceptable sensitivity, excellent positive predictive value, and good concurrent validity (Boyd et al., 2005).
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EPDS. The Edinburgh Postnatal Depression Scale (EPDS; Cox, Holden, & Sagovsky, 1987) is reportedly the most widely used screening questionnaire for postpartum depression, as it has been translated and used in diverse languages, cultures, and settings. The EPDS is a 10-item Likert-type self-report measure that purposefully assesses emotional and cognitive symptoms of depression, while excluding somatic symptoms, except for one item related to sleep difficulties, due to the normality of experiencing these symptoms shortly after giving birth. Systematic reviews of the EPDS have shown that the measure has moderate to good reliability, with a positive predictive value that is estimated to be -50% and a wide range of validity, sensitivity, and specificity values reported since the scales initial publication (Boyd et al., 2005). When using a clinical cutoff score > 12, the authors of the EPDS reported the scales sensitivity at 86% and the specificity at 78% (Cox et al., 1987). However, despite its widespread use as a PPD screening questionnaire, the EPDS includes symptoms of anxiety (Brouwers, van Baar, & Pop, 2001), and, therefore, interpreting results as a pure measure of PPD has been cautioned (Boyd et al., 2005).
PDSS. The Postpartum Depression Screening Scale (PDSS; Beck & Gable, 2000) is a 35-item Likert-type self-report instrument, assessing symptomatology within seven distinct dimensions (i.e. sleep and appetite disturbance, anxiety/insecurity, emotional lability, cognitive impairment, loss of self-esteem, guilt/shame, and suicidal thoughts), that has been validated for detecting the presence of depression in the postpartum period. The PDSS boasts 94% sensitivity and 98% specificity at detecting major depression in the postpartum period when employing a cut-off score of 80, and 91% sensitivity and 72% specificity at detecting major or minor depression in the postpartum period when employing a cut-off score of 60 (C. T. Beck & Gable, 2001a, 2001b). Additionally, the PDSS has been found to be valid for use
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with individuals of varied cultural and ethnic backgrounds (Zubaran, Schumacher, Roxo, & Foresti, 2010), and the measures 7th grade reading level may increase the likelihood that women of limited education are able to accurately complete the questionnaire (Boyd et al., 2005).
SCID. The Structured Clinical Interview for DSM-IV-TR (SCID; First, Spitzer, Gibbon, & Williams, 2002) is a diagnostic interview intended to specify patients that meet criteria for major mental disorders (i.e. Axis I disorders) according to the DSM-IV-TR (American Psychiatric Association, 2000). Several studies have demonstrated that the SCID has good reliability, with Kappa values ranging from 0.66 0.93 for diagnosis of major depressive disorder (Lobbestael, Leurgans, & Arntz, 2010; Skre, Onstad, Torgersen, & Kringlen, 1991; Williams et al., 1992; Zanarini et al., 2000; Zanarini & Frankenburg, 2001). The true validity of the SCID is difficult to ascertain, as the diagnosis of mental illness is largely subjective in nature, depending on the symptoms endorsed by the patient and potentially confirmatory observations made by the clinician or witness. However, the SCID was developed in an attempt to standardize the process of clinical diagnosis made ideographically by trained professionals, and, thus, the SCID is often referenced as the gold standard as to which diagnostic procedures are compared (Shear et al., 2000; Steiner, Tebes, Sledge, & Walker, 1995).
Assessment of Emotional Availability: The Emotional Availability Scales, 4th Edition (EAS; Biringen, Robinson, & Emde, 1998; Biringen, 2008) describe and assess six dimensions of dyadic interaction, with four on the adult side Sensitivity, Structuring, Non-Intrusiveness, and Non-Hostility, and two on the child side Responsiveness to adult and Involvement of
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adult. An asset of the EAS is that two dimensions measure child qualitieschild responsiveness to the caregiver and the childs involvement with the caregiver, thus capturing not only the adults side of the relationship, but also the childs side of the relationship. Table 2 briefly describes each domain, with corresponding subscales, included within the EAS, and Appendix D provides a more detailed review of the EAS scales and subscales. Although culture affects how parents and children interact with one another, the emphasis on the global presence of an emotional dyadic connection makes this tool an important one, even in varying cultural contexts. Co-investigators who received training and certification by the developers of the EAS rated previously recorded free play sessions that lasted 10 minutes. Co-investigators completing the EAS coding were blind with respect to maternal self-reports of symptoms of depression, clinical diagnosis, and OXTR genotype at the time of assessment and scoring.
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Table 2. Emotional Availability Scales and subscale descriptions
ADULT Sensitivity accurate reading of cues and expressing a range of emotions to establish a healthy connection
-Affect looks at range from balanced and genuine to bland or depressed/withdrawn (1-7) -Clarity of Perceptions awareness of signals and responds appropriately (1-7) - Timing Aware of importance of timing (1-3) -Flexibility Play is fun and adult is flexible (1-3) -Acceptance speaks/acts in respectful ways (1-3) -Amount of interaction right amount or too little (1-3) - Conflict moves conflicts to resolution (1-3)
Structuring Positive suggestions and guidance of child
- Guidance proactive and appropriate variety of suggestions (1-7) - Success successful attempts of moving child to higher level (1-7) -Amount Right amount of structure or too little/none (1-3) -Limit Setting appropriate boundaries are set (1-3) -Firm in Pressure can remain firm but connected in interaction, does not cave easily (1-3) - (Non)Verbal Structuring both, one, or neither channel used (1-3) -Peer vs. Adult Adult is clearly older and wiser (1-3)
Non-Intrusiveness Being available without being intrusive
-Following Child Lead has an appropriate spacious quality (1-7) -Ports of Entry waits for optimal breaks to enter interaction (1-7) Commands Dos used sparingly (1-3) -Adult Talking talking used as dialogue and not overpowering (1-3) -Didactic Teaching teaches without ignoring relating (1-3) -Interferences Only verbal and only when necessary (1-3) -Feel Intrusive child does or does not indicate the adult is intrusive (1-3)
Non-Hostility Displays of overt and/or covert hostility
- Lacks Negativity in words or tone of voice (1-7) - Lacks Ridiculing mocking/ridiculing statements or behaviors (1-7) - Lacks Threat of Separation use of separation as a threat (1-3) -Lose Cool ability to maintain composure during stressful time (1-3) -Frightening evidence of physical or verbal assaultive behavior (1-3) - Silent amount of silence in interaction (1-3) -Themes play is appropriate but not malevolent (1-3)
CHILD Child Responsiveness whether child responds to adult in positive way
-Affect ranging from upbeat to inappropriate to sad/irritable (1-7) Responsiveness when adult initiates, child responds (1-7) -Autonomy right amount of age-appropriate seeking (1-3) -Physical Positioning seeks or avoids physical contact (1-3) -Role Reversal parent-like behaviors by child (1-3) -Lack of Avoidance attentive to play and does not exclude (1-3) -Task Oriented appropriate level of focus on object play (1-3)
Child Involvement degree to which child involves the adult
-Elaborative Initiative engages in elaborate way (1-7) - Simple Initiative child initiates the engagement (1-7) -Use of Adult Uses adult for emotional exchange and not as a tool (1-3) - Lack of Over-Involvement negative or over-involving behaviors (1-3) -Eye Contact appropriate amount to none (1-3) -Body Positioning child positions body toward adult (1-3) -Verbal Involvement involves through talking/babbling (1-3)
Note: EAS domains listed in the shaded row, with domain specific subscales immediately following. Direct scores per domain consist of overall impression of subscales with scores ranging from 1-7. Eligible individual subscale scores included in parentheses.
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Assessment of Infant Development. The Bayley Scales of Infant and Toddler Development-Ill (BSID-III; Bayley, 2005; 2006) was used to assess infant cognitive, language, and social-emotional development (the motor and adaptive behavior scales were not administered). The items on the Cognitive Scale (Cog) of the BSID-III assess sensorimotor development, exploration and manipulation, object relatedness, concept formation, memory and other cognitive abilities. The Language Scale evaluates Receptive Communication (RC) and Expressive Communication (EC). The RC scale assesses auditory acuity, as well as preverbal behaviors related to vocabulary development, including being able to identify objects and pictures, and otherwise respond to words (Bayley, 2006). The EC scale assesses the childs ability to vocalize, and includes items on babbling, gesturing, joint referencing, turn-taking, first words, and object/picture naming. In addition, mothers were asked to fill out the Bayley Social-Emotional Scale (SE), which for the studys age range has 17 items designed to assess how well the child has attained social-emotional milestones, including self-regulation, positive and negative affect, activity level, adaptability to change, distractibility, and fear/anxiety (Bayley, 2006). The BSID-III exhibits good reliability, with reliability coefficients ranging from .74 to .92 for the Cog, RC, and EC scales in this studys targeted age group. Scale convergence and discriminant validity are also reported to be high (Bayley, 2006). Clinical psychology doctoral students who received training and supervision by an experienced, licensed clinical child psychologist administered the BSID-III scales. BSID-III administration was recorded, and administration of the Cog, RC, EC, and SE scales generally took between 30 and 60 minutes to complete. BSID-III administrators were also blind with
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respect to clinical diagnoses and maternal self-reports of symptoms of depression at the time of assessment and scoring.
Data Analysis Plan & Power
Data was analyzed using IBM SPSS Statistics version 24 (IBM Corp., 2016). Descriptive statistics (e.g. means, standard deviations, frequency distributions) were calculated to describe to the sample. Differences in demographic, genotypic, and clinical characteristics were assessed using an independent samples T-test as reported in Table 1.
The initial hypotheses, i.e. that there is a main effect of OXTR genotypes on maternal depression categorization, observed dyadic emotional availability, and infant cognitive development score, were tested using between factors analysis of variance (ANOVA). The broadest hypothesis looked at dyadic emotional availability in the presence of shared OXTR genotype pairings. A recent study found that in a representative sample of the USA population (n=348), 51.5% of the individuals carried a G/G genotype, 41.4% carried the A/G genotype, and 7.1% carried an A/A genotype (Poulin, Holman, & Buffone, 2012). Given the low overall presence of homozygous A OXTR genotypes in the general population and the overall positive finding for prosocial characteristics attributed to homozygous G genotypes, categorization of OXTR genotypes into two groups (i.e. those that are homozygous G and those carrying an A allele) were collapsed during posthoc analyses. However, initially, ANOVA analyses tested all possible genotypic pairings to determine underlying significance. With the 3x3 ANOVA structure, the studys 61 dyads obtained a power >78 with an effect size of .4 (Faul, Erdfelder, Buchner, & Lang, 2009). Data collection for 61 mother-infant dyads represents an acceptable sample size for statistical analysis to detect a large effect.
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Remaining hypotheses tested through additional 3x1 ANOVA analyses had a power >.78, based on the studys sample size (Faul et al., 2009).
Figure 3. Power based on sample size for a 3x3 ANOVA
F tests ANOVA: Fixed effects, special, main effects and interactions
Numerator df = 2. Number of groups = 3. a err prob = 0.05. Effect size f = 0.4
When an ANOVA indicated a statistically significant result, an analysis of covariance (ANCOVA) or multiple analysis of covariance (MANCOVA) was utilized to determine if the significant result remained after taking into account covariates (e.g. minority status, maternal education, mother/infant genotype) or shared variance amongst dependent variables.
Ethical Considerations
The Colorado Multiple Institutional Review Board (COMIRB) approved the research study protocol, consent forms, HIPAA forms, outcome measures, and advertisement materials. Appendix E provides the current and past COMIRB approval documentation for this study (COMIRB protocol #14-0851). In addition to protocol approval, COMIRB required that the principal investigator, as well as all study related personnel, satisfy the basic education requirements for human subjects research. All researchers involved in this study completed the CITI Basic course in human subject protections before taking part in any study related activities.
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Risks related to this study protocol were minimal given the non-invasive procedures utilized for data collection, and relatively large base of literature investigating OXTR genotypes with no evidence found for adverse outcomes. DNA was collected by obtaining buccal cells from saliva and all study materials were de-identified and given a unique study identification number prior to analysis. Participants chose to take part in the study and were allowed to stop at any time, but all participants completed the study after being briefed on the study and supplying informed consent. All study related materials could be destroyed if requested by the participant at any time during or after their participation in the study, but this was never requested by a participant. All information collected as part of this study took place with the informed consent and cooperation of the participant, and mothers were required to sign the consent authorization prior to infant saliva collection. No prior medical records or previous protected health information was requested as part of this study.
Additionally, all study related materials were de-identified and coded with a unique study identifier. De-identified data was retained in a password protected spreadsheet (i.e. SPSS file). All subject data collected in paper form was kept in a locked file cabinet in the office of the principal investigator. Videotape recordings of the 10-minute free play session were retained digitally, but deidentified and coded with the participants corresponding study identifier. Digital videotape recordings were stored in a password-protected folder on a restricted hard drive.
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CHAPTER III
RESULTS
Hypothesis 1: Genotype x Depression
The presence of depression in the postpartum period will be related to the presence of the G/G OXTR allele sequence in the mother.
Multiple one-way ANOVAs were used to test the initial hypothesis that a homozygous G genotype is related to increased rates of depression. As reported in Table 3, no significant differences were found between maternal genotype (i.e. GG, GA, or AA) and maternal depression diagnosis as identified by the SCID (n=56; p=.356), global assessment of functioning as reported on the SCID (n=59; p=.598), or depressive screening score on the BDI-II (n=59; p= 454), PDSS (n=60; p= 242) and EPDS (n=59; p= 314). When maternal genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozygous G genotype, maternal depression diagnosis was still not significant based on the results of a Pearson Chi-Square test (X2(3)=2.913, p=.405).
However, further posthoc analysis revealed that there was a significant effect on maternal depression by infant genotype (Table 3). A Pearson Chi-Square test of independence was performed to examine the relation between maternal depression screening score and infant genotype. The relation between maternal depression symptoms and infant genotype was significant (X2(i)=4.657, p=.031), with mothers of infants with a homozygous G genotype more likely to have higher depression screening scores according to the EPDS, but not for the BDI-II or PDSS. Further analysis revealed that there was also a significant difference between maternal depression diagnosis on the SCID and infant genotype as determined by a one-way ANOVA (F(2,52) = 4.434, p = .017). A Tukey post hoc test revealed that mothers of infants with a homozygous G genotype were significantly more likely to have
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a diagnosis of major depressive disorder than mothers of infants with a heterozygous genotype (p=.012). Maternal depression diagnosis was not significantly different based on the presence of an infant with a homozygous A genotype, though, this could be due to the low number of infants included in the sample with a homozygous A genotype (n=4). When infant genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozygous G genotype, maternal depression diagnosis was still significantly more likely to be observed, based on the results of Pearson Chi-Square test, when infant OXTR genotype was GG (X2(3)=8.440, p=.038). Not surprisingly, based on the maternal depression results highlighted above, current maternal GAF was also found to be related to infant genotype, such that mothers of infants with an A OXTR allele tended to have higher interviewer-rated GAF scores (r=.335, n=58, p=.010).
Table 3. Depression results by mother and infant genotype
Mothers Genotype GG GA AA GG GA/AA
Depression Screen
BDI-II score 12.3 (9.8) 10.2(6.8) 8.4 (3.8) 12.3 (9.8) 9.7 (6.1)
PDSS score 63.1 (21.5) 68.3 (25.0) 51.9 (13.3) 63.1 (21.5) 64.0 (22.2)
EPDS score 6.8 (5.6) 6.1 (4.1) 3.7 (1.7) 6.8 (5.6) 5.5 (3.78)
SCID Depression Dx
No Depression 10 (32.3%) 6 (30.0%) 3 (60.0%) 10 (32.3%) 9 (36.0%)
Full Remission 14 (45.2%) 12 (60.0%) 2 (40.0%) 14 (45.2%) 14 (56.0%)
Partial Remission 3 (9.7%) 0 (0.0%) 0 (0.0%) 3 (9.7%) 0 (0.0%)
Current Depression 4 (12.9%) 2 (10.0%) 0 (0.0%) 4 (12.9%) 2 (8.0%)
GAF rating 74.2 (11.4) 76.2 (11.0) 78.8 (11.9) 74.2 (11.4) 76.8 (11.1)
Infants Genotype GG GA AA GG GA/AA
Depression Screen
BDI-II score 13.2 (10.2) 9.2 (5.6) 7.7 (3.1) 13.2 (10.2) 9.0 (5.3)
PDSS score 67.3 (26.5) 58.7 (16.6) 65.3 (13.1) 67.3 (26.5) 59.6(16.1)
EPDS score 7.7 (5.7) 4.8 (3.3) 4.3 (2.5) 7.7 (5.7)* 4.75 (3.2)
SCID Depression Dx
No Depression 6 (20.7%) 13 (56.5%) 0 (0.0%) 6 (20.7%) 13 (50.0%)
Full Remission 15 (51.7%) 9(39.1%) 3 (100%) 15 (51.7%) 12 (46.2%)
Partial Remission 3 (10.3%) 0 (0.0%) 0 (0.0%) 3 (10.3%) 0 (0.0%)
Current Depression 5 (17.2%)* 1 (4.3%) 0 (0.0%) 5 (17.2%)* 1 (3.8%)
GAF rating 71.9 (11.1) 80.5 (10.4) 73.3 (7.9) 71.9 (11.1) 79.4 (10.3)*
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Note: Columns are defined based on Genotype. Last two columns compare homozygous G carriers with carriers of a minor allele. Numbers in parentheses are standard deviations, unless denoted as a percentage of the sample.
* denotes p<05. Abbreviations: Beck Depression Inventory, 2nd Edition (BDI-II), Postpartum Depression Screening Scale (PDSS), Edinburgh Postnatal Depression Scale (EPDS), Structured Clinical Interview for DSM-IV-TR (SCID), Diagnosis (Dx), GAF = Global Assessment of Functioning (GAF), as estimated from SCID interviews.
Hypothesis 2: Genotype x EAS
Mothers carrying an A OXTR genotype will demonstrate decreased levels of Sensitivity as measured by the Emotional Availability Scales.
A one-way ANOVA did not find any significant differences between maternal genotype and direct score of Sensitivity on the dyadic Emotional Availability Scales (F(2,57) = 1.223, p = .302). Additionally, a separate one-way ANOVA did not find any significant differences between infant genotype and direct score of Sensitivity on the EAS (F(2,56) = 1.146, p = .325). However, further review of the EAS demonstrated a significant difference between infant genotype and direct score of Non-Intrusiveness (F(2,56) = 4.034, p = .023), such that mothers of infants with an A OXTR allele tended to score higher (i.e., closer to optimal), on the Non-Intrusiveness scale (r=.268, n=59, p=.040).
Table 4. EAS correlations of direct scores
Sensitivity Structuring Non- Intrusiveness Non- Hostility Child Responsiveness Child Involvement
Sensitivity 1
Structuring .731** 1
Non-Intrusiveness .385** 0.225 1
Non-Hostility .503** .581** 0.174 1
Child Responsiveness .375** .427** .316* .286* 1
Child Involvement .578** .530** .387** .410** .637** 1
Note: ** denotes significance at the 0.01 level (2-tailed), and denotes significance at the 0.05 level (2-tailed).
However, due to concerns with multicollinearity of the EAS direct scale scores (Table 4), a posthoc multivariate analysis of variance (MANOVA) was performed with each of the
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direct scale scores of the EAS entered as a dependent variable and infant and mother genotypes entered as fixed factors (i.e. GG vs GA/AA). The results of the MANOVA still demonstrated a significant difference between infant genotype and direct score of Non-Intrusiveness on the EAS (F(l,58) = 5.779, p = .020, r|2=.097). However, as shown in Table 5, when minority status and maternal education were entered into a multivariate analysis of covariance (MANCOVA) infant genotype failed to show significance related to the Non-Intrusiveness direct scale score, but maternal education was significant (F(l,59) = 14.623, p = .000, r|2=.219). This suggests that the variance in mean scores of Non-Intrusiveness direct scale scores is best accounted for by maternal education, as opposed to infant genotype.
Table 5. Non-Intrusiveness direct score MANCOVA result
Sum of Squares df Mean Square F Partial Eta Squared
Maternal Genotype 3.865 1 3.865 3.712 .067
Infant Genotype 3.188 1 3.188 3.062 .056
Minority 1.661 1 1.661 1.595 .030
Maternal Education 15.225 1 15.225 14.623* .219
Note: denotes p=.000.
Hypothesis 3: Genotype x Infant Performance
Infants of depressed mothers who are carriers of an A OXER allele inherit a protective genetic factor that leads to more optimal performance on the Child Responsiveness direct score on the EAS and cognitive development score on the BSID-III.
Ratings of Child Responsiveness on the Emotional Availability Scales. A one-way ANOVA did not find any significant differences between infant genotype and direct score of Child Responsiveness on the EAS (F(2,56) = 1.359, p = .265). Additionally, after collapsing infant genotype groups to differentiate those that were carriers of an A allele from those that were homozygous G genotype, direct scores of Child Responsiveness on the EAS was still not significant based on the results of a Pearson Chi-Square test (X2(6)=7.522, p=.275).
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In posthoc analysis, the Child Responsiveness total, direct, and subscales scores were entered into a MANCOVA that tested associations between mother and infant genotypes and demographic covariates. The total score, direct score, and subscale scores (i.e. Affect, Responsiveness, Autonomy, Physical Positioning, Lack of Avoidance, and Task Oriented) were entered as dependent variables and infant/mother genotypes and concordance were entered as fixed factors, while minority status and maternal education were entered as covariates. The results of the MANCOVA demonstrated a significant difference between maternal genotype and Responsiveness subscale score on the EAS (F(l,59) = 3.984, p = .051, r|2=.071), such that mothers that carried an A allele were associated with marginally higher mean (i.e. GA/AA x = 5.919; GGx = 5.617) scores as observed on the Responsiveness subscale of the Child Responsiveness domain. Additionally, as a covariate higher maternal education significantly differentiated observed scores on the Child Responsiveness total score (F(l,59) = 4.690, p = .035, r|2=.083), Autonomy subscale score (F(l,59) = 5.058, p = .029, r|2=.089), and Physical Positioning subscale score (F(l,59) = 4.036, p = .050, r|2=.072).
Cognitive development, as measured by the Bayley s Scales of Infant Development,
3rd Edition. A one-way ANOVA did not find any significant differences between infant genotype and cognitive development scaled score on the BSID-III (F(2,53) = .141, p = .869). Additionally, after collapsing infant genotype groups to differentiate those that were carriers of an A allele from those that were homozygous G genotype, no significant differences were found on the cognitive development scaled score based on the results of a Pearson Chi-Square test (X2(9)=4.009, p=.911). Figure 4 shows the mean BSID-III cognitive development percentile rank by infant genotype.
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Figure 4. BSID-III percentile rank by genotype
BSID-III percentile rank by genotype
I Maternal GG H Maternal GA/AA Infant GG S Infant G A/A A Concordant Not Concordant
80%
72% 71%
70% 68% 68% ^ 68%
72%
60%
50%
40%
30%
20%
10%
0%
!
!
52% 51/
54% 54%
Cognitive
52% 53%
1-1
1
Receptive Communication Expressive Communication
Note: BSID-III average percentile ranks shown by genotype for Cognitive, Receptive Communication, and Expressive Communication domains. No significant associations were found between mother, infant, or concordant genotypes and BSID-III scaled scores.
Posthoc analysis considered infant and maternal genotype in relation to not only the cognitive development scale of the BSID-III, but also the expressive and receptive communication scales. The cognitive, expressive communication, and receptive communication scaled scores were entered as dependent variables and infant and mother genotypes were entered as fixed factors (i.e. GG vs GA/AA), while minority status and maternal education were entered as covariates. The results of the MANCOVA demonstrated
that, in our sample, minority status accounted for a significant portion of the variance recorded on the BSID-III receptive communication scale (F(l,56) = 4.522, p = .039), but not for the expressive communication scale (F(l,56) = .508, p = .479) or the cognitive development scale (F(l,56) = 1.106, p = .298). No significant differences were found in relation to either maternal or infant genotype in regards to the BSID-III scaled scores for the cognitive, expressive communication, or receptive communication scales (Figure 4).
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Hypothesis 4: Concordance x EAS
Non-depressed mother-chdd dyads that are both carriers of the homozygous G genotype will demonstrate significantly higher dyadic emotional availability ratings than dyads that did not meet this criterion
Repeated Pearson Chi-Square tests showed that there were no significant differences between maternal depression diagnosis and concordant homozygous G genotype with any of the direct scores on the EAS: Sensitivity (X2(5)=3.736, p=.588), Structuring (X2(6)=2.651, p=.851), Non-Intrusiveness (X2(6)= 577, p=.997), Non-Hostility (X2(4)=.867, p=.929), Child Responsiveness (X2(6)=8.75, p=.188), or Child Involvement (X2(7)=5.792, p=.564).
In posthoc analysis, concordance was extrapolated to not only include mother-infant dyads that had a homozygous G genotype but rather any concordant dyadic genotypes (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant), and compared to the results of the EAS. A MANOVA was first conducted to determine if genotype concordance was associated to any of the six direct scores on the EAS. Genotype concordance was not statistically associated with the observed variance in mean scores on the Sensitivity, Structuring, Non-Intrusiveness, Non-Hostility, Child Responsiveness, or Child Interaction direct scores included in the EAS.
Although the EASs Sensitivity direct score seemingly maps onto the earlier reported finding between maternal OXTR and sensitivity reported by Bakermans-Kranenburg &
Van Ijzendoom (2008), it should be noted that this prior studys measure of sensitivity was based on observer ratings in three distinct subscales (i.e. supportive presence, intrusiveness, and clarity of instruction) that drew from Ainsworths conceptualization of social responsiveness (Ainsworth, C., Waters, & Wall, 1978; Egeland, B., Erickson, Clemenhagen-Moon, Hiester, & Korfmacher, 1990). Therefore, a separate analysis of the subscales included within the EASs Sensitivity construct looked at mean differences in rater scores
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based on maternal genotype, infant genotype, and concordant genotypes. No significant differences were found for either maternal genotype or infant genotype on any of the seven distinct subscales of the Sensitivity domain on the EAS. However, a between-subjects analysis of covariance (ANCOVA) revealed that, for the Affect subscale of the Sensitivity construct, OXTR genotype concordance accounted for a significant proportion of the variance in raters Sensitivity Affect score after minority status, maternal education, and mothers and infants genotypes had been separately taken into account (F(l,54)=7.167, p=.010, r|2=. 119; Figure 5). Additionally, a separate one-way ANCOVA demonstrated a statistically significant difference between OXTR genotype concordance on the Interaction subscale of the Sensitivity construct after controlling for minority status, maternal education, and mothers and infants genotypes (F(l,54)=6.193, p=.016, r|2=. 105; Figure 5). Furthermore, these results held when entering all Sensitivity scale based scores into a MANCOVA to rule out the potential effect of shared variance between scale/subscale scores.
For posterity, additional posthoc MANCOVAs were run for each of the remaining EAS domains, with their respective subscales entered as dependent variables. No significant associations were made between OXTR genotype concordance and the subscales of the Structuring, Non-Hostility, Child Responsiveness, or Child Interaction domains of the EAS. However, after minority status and maternal education had been separately controlled for, OXTR genotype concordance accounted for a significant proportion of the variance in raters Non-Intrusiveness Following Child Lead subscale score (F(l,54)=4.246, p=.044, r|2=.072), and in raters Non-Intrusiveness Adult Talking subscale score (F(l,54)=5.767, p=.020, r|2=.095). In each of the reported findings associating dyadic concordance of genotypes to subscales scores of the Sensitivity and Non-Intrusiveness domains, concordance related to
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higher scores on the subscales (Figure 5).
Figure 5. Concordance by significant EAS subscale
6.556
OXTR concordance by EAS subscale mean score
Concordant Not Concordant
6.000
Affect
2.861
2.609
Interaction
Sensitivity
6.069
5.848
Child Lead
2.806
2.565
Adult Talk
Nonlntrusiveness
Note:
Mean scores shown by genotype concordance for Sensitivity Affect, Sensitivity -Interaction, Non-Intrusiveness Adult Talk, and Non-Intrusiveness Follow Child Lead EAS subscales. Sensitivity Affect and Non-Intrusiveness Follow Child Lead subscales scored on a 7-point scale, Sensitivity Interaction and Non-Intrusiveness Adult Talking subscales scored on a 3-point scale. OXTR concordance was associated with increased subscale mean scores for each of the displayed scales (p<.05), after controlling for minority status and maternal education.
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CHAPTER IV
DISCUSSION
Oxytocins purported relationship with depression, prosocial parenting behaviors, and constructs such as empathy, trust, and love make it an important variable to study within the mother-infant dyad. This study was designed to determine the OXTR rs53576 genotype for a population of mother-child dyads, and then analyze the results in relation to measures of maternal depression, infant development, and dyadic emotional availability. In this way, the study was able to tests some previously reported findings regarding the effects of maternal genotype, while also going a step further to look at the impact of genotypic concordance within mother-infant dyads. The studys hypotheses were formulated based on empirical reports suggesting that the G allele, and in particular homozygous G genotypes, predicts decreased sensitivity to stress, improved social skills, prosocial parenting skills, and increased empathy (Bakermans-Kranenburg & Van Ijzendoom, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010). Provided the above tenants held true for our sample, it was believed that the benefits of prosocial parenting behaviors, decreased stress reactivity, and increased love, trust, and empathy would provide for a more optimal dyadic emotional availability between mothers and infants. Moreover, it was believed that optimal dyadic emotional availability would provide appropriate and timely scaffolding for infant development (Vygotsky, 1962, 1978).
However, several reports have also suggested that there may be a relationship between the homozygous G genotype and depression. For instance, there is a possibility that a homozygous G genotype may confer a susceptibility for depression (McQuaid et al., 2013;
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R. J. Thompson et al., 2011) and depressive symptomatology may suppress the positive effects associated with the homozygous G genotype (Riem et al., 2011). Additionally, the presence of a G allele may increase the likelihood of offspring developing major depression themselves if raised by a depressed caregiver (R. J. Thompson et al., 2011). Given the high prevalence of pregnancy related depression (Gavin & Gaynes, 2005), this studys sample may provide insight into OXTR rs53576s role in maternal depression, dyadic emotional availability, and infant development.
Null Hypothesis Results
The studies main hypotheses concerning links between OXTR rs 53576 variants and (1) maternal depression, (2) maternal dyadic sensitivity (3) infant performance on the EAS and BSID-III, and (4) observed EAS scores in relation to dyadic genotype pairings and maternal depression were not supported by these data. Maternal OXTR genotype was not associated with increased rates of depression, as measured by a common PPD screening measure or through a structured clinical interview. This finding may be an artifact of the particular postpartum study sample or the limited sample size, however, it also casts doubt on to the reliability of a previously reported finding suggesting that the presence of a particular OXTR allele predicts increased rates of depression (McQuaid et al., 2013; Saphire-bernstein et al., 2011), while supporting more recent meta-analytic findings raising questions about the association between OXTR rs53576 and psychopathology (Bakermans-Kranenburg & van Ijzendoorn, 2014; Li et al., 2015). Within a biopsychosocial context (Engel, 1977), genotypic data only partially accounts for biological factors and do not inherently consider the impact psychological functioning and social environment contribute to the development of psychopathology.
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The second hypothesis tested whether the predicted negative effects related to the presence of an A OXTR allele were supported with respect to the quality of Sensitivity observed within dyadic interactions as measured by the Emotional Availability Scales, 4th Edition (Biringen, 2000). Neither maternal nor infant genotype alone accounted for a significant difference in total or direct scores of dyadic Sensitivity. Due to the observational nature of the EAS and the fact that mother-infant interactions took place within a laboratory setting, it is possible that the assigned scores did not accurately represent the mother-infant dyads true emotional availability. It is also possible that the brief laboratory interactions we used to code emotional availability did not accurately reflect the norm for these dyads. Such that the setting and nature of the study may have inherently pulled for mothers to demonstrate what they believed to be prosocial parenting behaviors and optimal dyadic emotional availability. However, it might again be the case that looking at a single OXTR genotype is too reductionistic to explain the complex intricacies enmeshed in dyadic emotional availability between mothers and their infants.
The third hypothesis attempted to test the idea that infants/children carrying an A allele may inherit a protective factor related to social competence, empathy, and proneness to psychopathology as hypothesized by Feldman ( 2012c). Within the context of this study, the potential protective factor was operationalized through Child Responsiveness, as measured by the EAS, and infant cognitive development, as measured by the BSID-III. Tests revealed no statistical difference as a function of infant genotype on either observed Child Responsiveness or on the results of the cognitive scale of the BSID-III. It may still be true that the A allele provide[s] a buffer that mitigates some of the effects of maternal depression (Feldman, 2012b, p. 387), but as operationalized for this study infant genotype
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did not predict improved responsiveness or cognitive development in the presence of maternal depression. Moreover, this null finding did not offer support for an early difference in child phenotype based on OXTR genotype, as has been reported for adolescents growing up with a depressed mother (S. M. Thompson et al., 2014).
The fourth, and final, hypothesis attempted to investigate the role OXTR genotype concordance may have in differentiating observed dyadic emotional availability, when mothers are not depressed. Given the purported positive associations with parenting behavior and child social competence, it was believed that mother-infant dyads sharing a homozygous G genotype would be observed to provide more optimal interactions. However, in our sample, genotype concordance did not significantly discriminate between mother-infant dyads observed performance on the emotional availability scales. It is important to note that only the direct scores for the six emotional availability constructs were included in this hypothesis original analysis, and as previously discussed the mother-infants brief 10-minute videotaped interactions were recorded after being provided informed consent while in a laboratory setting. It is possible that dyadic interactions lasting longer in duration or taking place in a more natural environment may provide different results.
Post Hoc Analysis
Potentially the most noteworthy findings to emerge from this study occurred after rejecting the initial hypotheses, and then further investigating the role dyadic OXTR genotypes relate to measures of depression and emotional availability. As reported under the results for hypothesis #1 (Genotype x Depression), several unique findings related to infant OXTR genotype were related to maternal depression. For this studys sample, it appears that maternal depression status was related to the infants genotype using both a standardized
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depression screener and a structured clinical interview. The results demonstrate that mothers, regardless of their own genotype, are more likely to report symptoms of major depression when their infant had a homozygous G OXTR genotype. Given the increased rates of depression, daily functioning, as measured by GAF scores, were understandably also reduced for the mothers of infants that were homozygous GG carriers. The inverse way to interpret this result would suggest that mothers of infants that carry an A OXTR allele are less likely to report symptoms of depression and demonstrate more optimal daily functioning.
Additional analyses investigating the role of infant OXTR genotype and dyadic genotype concordance also produced some notable results, as reported under the results for hypothesis #2 (Genotype x EAS). First, mothers were observed to score more favorably on the construct of Non-Intrusiveness when their child carried an A OXTR allele. Non-Intrusiveness refers to a mothers ability to be present to their child, both emotionally and physically, without being intrusive (Biringen, 2008). Inherent in this conceptualization of nonintrusive parental behavior is a dependency on an infants reactions to their mothers actions and cues. In this way, the findings demonstrate a potential benefit in dyadic interactions based on infants carrying an A OXTR allele. However, this significant result became nonsignificant when maternal education was entered as a covariate. Still, it is notable that we again see the emergence of a potential benefit for dyads whose infants carry an A OXTR allele.
Another notable finding was discovered while conducting posthoc analysis related to hypothesis #3 (Genotype x Infant Performance). When considering maternal genotypes relation to observed scores on the subscales of the Child Responsiveness domain, mothers that carried an A allele were associated with an infants higher mean scores as observed on
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the Responsiveness subscale of the Child Responsiveness domain. According to the EAS, Child Responsiveness refers to the infants reaction to parental bids for engagement, and the expression of pleasure. The focus of this domain is on the emotional and social responsiveness displayed by the infant toward the mother. An optimally responsive child is one who is happy in the presence of the mother and is emotionally and behaviorally responsive. In fact, the developer of EAS states that the Child Responsiveness domain is closest to the attachment theory view of the secure/insecure child (Biringen, 2008). Within this domain, the Responsiveness subscale gauges the childs response to adult imitations. In this way, maternal carriers of an A allele in our sample were associated with more optimally responsive infants. This finding again suggests a potential benefit for the partner in a mother-infant dyad when their corresponding partner carries an OXTR A allele.
Furthermore, posthoc analysis looked at the relationship genotype concordance within mother-infant dyads had on observed EAS scores. The term concordance was used to refer to the sameness within genotypes collected from mothers and their infants (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant). Evidence was found while conducting additional analysis on hypothesis #4 (Concordance x EAS) supporting a role dyadic OXTR genotype concordance has on higher observed mean scores on the Affective and Interaction subscales of the Sensitivity domain and on the Following Child Lead and Adult Talk subscales of the Non-Intrusiveness domain on the EAS. As conceptualized by the Emotional Availability Scales, 4th Edition, the Affect and Interaction subscales are particularly salient to the construct of Sensitivity. The EAS manual states The key characteristic of the sensitivity construct, in our view is affect (Biringen, 2008, p.16) because The child also enjoys interactions with the adult. In other words, the adult cannot
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look good without the child (Biringen, 2008, p.17). Moreover, Non-Intrusiveness refers to the appropriateness of directedness on the part of the mother, providing balanced stimulation when interacting with her child. In an optimal non-intrusive relationship, there is a lack of over-directedness, over-stimulation, interference, and over-protectiveness. The Non-Intrusiveness scale holds a high respect for the autonomous pursuits of the child. Higher scores on Following the Child Lead and Adult Talk subscales of the Non-Intrusiveness domain captures the essence of the EASs conceptualization of an optimal non-intrusive dyadic relationship.
Empirical Significance
By testing the originally stated hypotheses and performing additional posthoc analysis, this study was able to provide additional evidence for OXTR rs53576 purported role in maternal depression, prosocial parenting or sensitivity, infant development, and dyadic emotional availability. Based on this studys significant findings, it is worth considering whether a mothers or infants genotype or the concordance of dyadic genotypes are partially responsible for driving reports of maternal depression or on observed measures of emotional availability. Klahr et al. (2014, 2015) highlighted the possible importance of evocative gene x environment interactions in interpreting gene associated studies. This studys findings offer tentative support for the idea that OXTR rs53576 genotype analyzed in isolation may not adequately account for outcomes within close social relationships. As highlighted in Feldmans recent review article on The Neurobiology of Human Attachments (2017, p.87), biobehavioral synchrony, the coordination of biological and behavioral processes between attachment partners during social contact, is a critical component of human attachments (Feldman, 2012a, 2015, 2016, 2017; Romero-Fernandez, Borroto-Escuela, Agnati, & Fuxe,
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2013). From this perspective, prior reports that investigated the role of maternal OXTR genotype on sensitivity, without consideration for the potential importance of dyadic concordance in their samples genotype pairings, may be due to a type II error if concordance was not collected or analyzed. For instance, it could be hypothesized that if the findings of this study regarding the importance of genotypic concordance on measures of Sensitivity on the EAS held true for other samples, then it is possible that Bakermans-Kranenburg & van Ijzendoorns often cited finding linking the maternal GG genotype with increased sensitivity scores (Bakermans-Kranenburg & Van Ijzendoorn, 2008) may vanish. Which, in turn, would inadvertently support Li et al.s (2015) meta-analysis disassociating a link between OXTR rs53576 polymorphism and sociability in the context of close relationships. Alternatively, the results of the two recent OXTR related meta-analyses reporting on associations between genotype polymorphism, psychopathology, and sociability may be quite different if concordant genotype analysis was available.
Additionally, based on the results obtained in this study, it may be increasingly important to consider the infants or childs genotype in the assessment of prosocial parenting, emotional availability, and postpartum depression. In line with the recent conceptualization stressing the coordinated dyadic nature of optimal oxytocinergic system functioning, the results of this study demonstrate that an offsprings genotype may be related to more or less optimal maternal functioning. Considering the model of biobehavioral synchrony proposed by Feldman (2017) for initiating and maintaining human attachments, the findings associating child genotype and dyadic concordant genotype pairings with depression status and observed components of emotional availability, respectively, may
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illustrate a small facet of the complexity involved within the biobehavioral underpinnings of the endocrine and neurological systems.
It also should be noted that, within this study, significant findings suggest that the presence of an A allele may impact a partners depression symptomatology or observed dyadic emotional availability. In regards to depression status, mothers reported less symptoms and had more optimal daily functioning when their infants carried an A allele. Similarly, mothers were found to display more optimal dyadic emotional availability ratings on key subscales included within the Sensitivity and Non-Intrusiveness domains of the EAS when their infants carried an A OXTR allele. Moreover, we found that critical areas of a Childs Responsiveness within the context of dyadic emotional availability was rated more favorably when mothers themselves were carriers of an A allele. An interpretation of this finding may suggest that carriers of an A allele demonstrate increased autonomy which then signals a sense of well-being back to their partner further perpetuating more optimal oxytocinergic functioning within a biobehavioral synchrony framework. Additionally, these partner dependent findings may support the notion posited by Feldman (2012a) that the A allele confers a sort of protective factor.
Study Limitations
Despite the acceptable power of the current study, concern is warranted due to the relatively small sample size and dependence on a large observed effect. There is a chance that results of this study did not adequately describe the main effects for the greater population of mother-infant dyads in the US, particularly given that ethnic and cultural differences have been noted in the literature as a potential confound for OXTR related studies (Feldman et al., 2016). Additionally, parent characteristics may have influenced observer
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report of dyadic emotional availability and behavior (Pauli-Pott, Mertesacker, & Beckmann, 2005; Sameroff, Seifer, & Elias, 1982). However, prior studies have demonstrated that mothers tend to provide a valid report of their children (Bates & Bayles, 1984), and a prior study looking at the effects of OXT administration on paternal emotional availability has been published with a slightly smaller sample size (Naber et al., 2010). Also, despite the myriad of studies associating OXTR to human affiliation, prosocial behaviors, and psychopathology, effects are likely due to a complex biopsychosocial processes that relies on environmental contingencies and vast biological systems (Benarroch, 2013; Donaldson & Young, 2008; Feldman, 2017).
Conclusion
Oxytocin and its single receptor, OXTR, have been well studied since becoming the first human polypeptide hormone sequenced and biochemically synthesized. Oxytocins role in uterine contractions and mammalian milk production led to findings demonstrating the hormones role in pair bonding, social affiliation, and attachment. More recently, studies have demonstrated significant relationships between oxytocin levels and OXTR genotypes in relation to prosocial parenting behaviors, measures of empathy, trust, and love, as well as psychopathology. However, as we continue to learn more about the complex and multifaceted nature of attachment and social affiliation, researchers have begun to investigate the role biobehavioral synchrony plays. Mother-infant dyads are a particularly interesting sample to study these effects, as biopsychosocial factors directly impact interpersonal relations at a critical point in infant development and dyadic attachment. Results of this study suggest that maternal genotype alone may not be enough to discriminate predictors of complex dyadic interactions. Biobehavioral synchrony between both members of the dyad,
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and specifically OXTR genotype concordance, may offer increased insight into the role oxytocin and OXTR play in observable dyadic emotional availability, as well as the greater development of pair bonding, attachment, and social affiliation. In the same breath, given the complexity of biopsychosocial factors impacting observable and measureable outcomes of interpersonal interactions, it is important to continue to look at additional pathways that may work in parallel to form meaningful interpersonal connections. Moreover, future research should look to replicate previously published findings regarding OXTR rs53576s purported association with social affiliation, prosocial parenting, and psychopathology with particular attention on how phenotypes being studied are operationalized, as well as being mindful about how environmental, ecological, and cultural differences can confound study results.
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66


CONCORDANCE POSTER PRESENTATION
t"
VO
Does Concordance Matter? Analyzing Dyadic OXTR Polymorphism in Relation to Maternal Depression and Child Responsiveness
Ryan Asherin, Kevin Everhart, Jo Vogeli, Josh Fowler, Christopher Phiel, & Peter Kaplan
University of Colorado, Denver, USA
Highlights:
Prbr research has linked variations in a s'ngle nudeotde polymorphism (SNP) in the oxytocin receptorgene (OX IK _53576j with differences in maternal depression and maternal sensitivity.
We examined correlations between maternal and infant OXTRgene polymorphisms, maternal depression, maternal sensitkrity, and chid responsiveness, as well as effects of genotype concordance/discordance within the mother infant dyad.
Structured clinical I ntetview-based Global /Assessment of Functioning (GAP) in mothers was lower when both mother and child had the GG genotype, relative to all other combinations.
Infants responsiveness to maternal bids Ibr attention was lower in dyads with GG relatkretoAG'AA dyads but concordance did notmatter
Introduction:
Oxytocin (OT) is a neuropeptide synthesized in the hypotealamusthat functbns in parturition, nursing, parental care, and more general^, in promoting pro-social behavior (Bakemans-K'anenburg &van Jzendoorn, 2008). T he action of OT is thought tobe dependent on the eopression of its receptor, which is encoded by theoxytocin receptor gene(OXTR), located on chromosome 3p25. A angle nucleotide polymorphism (SNP) onthegenes third htron, OXTRj-s53576, with aguan'ne (G)-adenine (A)subiitu(on, has been linked to differences in maternal sensitivity and depressve symptoms, among other behavioral traits. Generally speaking, the A allele has been considered to be the risk alele, and is correlated wlh heightened sensitivity to stress, impaired social skils, poorer parenting, and non-optimal mental health outcomes. For example, AG and AA versus GG carriers have been reported to exhibit lower behavioral sensitivity wife their infants (Bakermans-Kranenburg & van IJ$ndoom, 2008). Hcwever, other research suggeststhat individuals with the GG genotype may be more aisceptible to depression (Costa et al., 2009) and rroresensitkretoadverselife eoperiences and lack of social support (Kim et al., 2010), possibly because these experiences differentially affect OXTR expression in GG individuals pell et al., 2015). In addition, ittle research has considered whether tee OXTR genotype of the infant interacts with that of the mother in predicting and maternal postpartum depression, maternal sensitivity, and infant responsiveness, although intent OXTR_rs53576 and otherSNFShave been studied and ruled out as meaningful predictors of mother-intent attachment (Roisman et al., 2013).
Methods:
Sixty-one mothers and their4- b 14-month old infants wee reciuited through a Facebook advertisement Table 1 summarizes demographic information by OXTR genotype. Using a dominance model, we combined A3 and AA carries and contrasted them with GG carriers. Mothers completed tee feck Depression Inventory, 2nd Edition (EDI-II) the Fbstpartum Depression Screening Scale (PDSS), and participated in a stiuctured clteical interview to determine their Global Assessment of Function'ng (GAF) rating. Ail dyads also partbipated in a 10-minute semi-structured play session, whichwas videotaped for later coding using the Emotional Availably Scales (EAS) Finally, tefants participated in a farriiarization-prefeience test in whbh they were shewn 1-minute videos of an unfamiliar woman speaking te intent-directed speech (IDS) or adult-directed speech (ADS) intonation. Duratbnsof look'ng at the video were recorded.
The EAS is agbbal coding scheme with four caregkrer scales (caregiver sensitivity, structuring, non-intrusiveness, andnon-hostiity) ancitwochib scales (responsiveness to caregiver attentional bids and involvement of the caregiver in play). Each major scale is also broken down inb subscales to characterize the details of that dimension.
DNAsamples according toll 3/DDK-SO) b<
aSTEPONE qPCR m
inGG andAG/AA rr
Functioning (GAF). GAF did not differ
Conclusions
Variations in the OXTR_rsS3S76 SNP in mothers of 4-to 14-month-old infar were not correlated overall with differences in maternal self-reported sympt
Global Assessmentof Functioning (GAF) rated from Structured Clinical Interviews (SCIDs). Although the A allele has often considered to be therist allele for depression,there is some evidence that GG individuals may be m attuned and sensitive to both positive and negative experiences,and perha
with a high level of methylation in OXTR_rs53576 exhibit increased sympto of depression relative to those vrith at least one Aallele (forwhom methylati lev el did notmatter; Bell et al., 2015).Early life stress,abuse,or neglectma be involved with this kind of epigenetic effect (McQuaid et al, 2013).
In contrastto some previous res earch,OXTR_53576 genotype was not significantly related to maternal sensitivity,but our sample size was small a
functioning ofGGGG dyac
:t, AG mothers'GAF ratings
2. Emotional Availability Scale
Matemal_0>ORjeno^£e.A 2x 6 MANOVA with maternal genotype (AG/AA v GG) as the independent variable and the 6 EAS scales as the dependent measures showed only a signilicanteffect on the Child Responsiveness scali F(1,55) = 3.94, p = .05, r|2= .09, with higher responsiveness in the children c AG/AA than GG mothers. Infant OXTR genotype was unrelated to EAS ratings
EMOTIONALAVAI LABILITY SCALE
So What?
These findings are c to be determined. Or
expression oftheox ytocin receptor in the s
leading to higheroxi rtoc in to notion in GG tt
OXTR_53576 SNP i s located on the gene':
portion of the gene. so exactly how it affect
receptor is still uncle ar.This SNP may con
may be involved dut - to linkage dis equilibri
polymorphisms.In a ny event, higher levels
the maternal brain''; and help mothers toco
Iransition (Bell et a I, 2015).This hypothes
GG individuals have :been characterized a
possibly more sensi live to environmentalir
early life adversity. ' be 'ess socially attorn
ariations in OXTR_53576 affect mygdala and other brain regior
5i53SEs3S

Acknowledgements:
af ColoradoDenve


APPENDIX B
ISOHELIX SWAB BROCHURE
DNA Buccal Swabs
DNA Stabilisation and Isolation Kits
i
ce//xJOOOC projects,
F
Isohelijj
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Each Isohelix product has been specifically designed and developed to improve all the DNA sampling steps from our original product; the DNA buccal swabs right through to the DNA isolation and even if you wish checking the DNA's PCR integrity. We have taken an integrated approach by looking at each individual step to improve yields, reduce sample losses and at the same time improve the handling procedures, the result maximises yields and quality of DNA.
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The Isohelix range of Buccal Swabs has been specifically designed to give increased yields of high quality buccal cell and genomic DNA. Buccal cell sampling offers a viable alternative to blood collection and, together with the Isohelix range of DNA Isolation and Stabilisation kits, enables high yields of pure, intact DNA to be collected easily and quickly. Isohelix swabs are suitable for both human and veterinary use and offer significant advantages over other swab designs in terms of efficiency of cell collection by the unique swab matrix, which combined with a quick release surface, maximises yields of DNA. Swab batches are ethylene oxide treated and routinely tested for human DNA contamination.
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68


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projects
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The SK-l swab uses the unique swab matrix design to efficiently collect buccal cell samples. Following sampling you simply snap the shaft just above the swab head and place into the tube provided, then seal for containment.
V Swab matrix significantly improves DNA yields S Various formats available with/without tubes
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Instructions for Isohelix SK-l, SK-3, SK-4 and SK-5 Buccal Swabs
U/^P Pull open the package from one end.
Remove one of the swabs from the tube.
Insert the swab Into your mouth and rub firmly against the inside of yourcheekor underneath lower or upper lip. For standard DNA collection rub for 1 minute and in all cases rub for a minimum of 20 seconds. Important use reasonable, firm and solid pressure
Place the swab back into the tube. Do not touch the brush with your fingers.
Place your thumbnail in the small groove set in the handle, then snap the handle in two by bending to one side. Let the swab head fall into the tube.
Seal the tube securely with one of the caps.


Buccal swab stabilisation
i
f AAAAA 1
projects i
Isohelix Dri-Capsules cat. no:sgc-so
Designed for use with Isohelix SK-1 swabs, these silica gel capsules offer an easy to use alternative to chemical stabilisation for long term stability of the buccal DNA on the swab head prior to isolation.
S Long term stability at room temperature S Liquid free alternative to DSK Kit S Easy to handle by patients and professionals S Designed for use with SK-1 swab packs S ideal for remote sample collection
Long term stability studies show that the stability of DNA samples on SKI swabs stored with Isohelix Dri-Capsules is maintained over a period of at least 3 years.
The study is ongoing and is expected to confirm an increased period of stability well in excess of the 3 years tested so far.
Isohelix DSK Stabilising and Lysis Kit
Cat. No: DSK-50
The Isohelix Stabilising and Lysis Kits fully stabilise your DNA from enzymatic and microbial activity that occurs naturally after buccal sampling. The kit is designed to store the samples at room temperature and shows no visible loss of stability for at least 5 years (tested to date). The DSK Kit fully lyses the cells, releasing additional DNA from the cells further enhancing yields, whilst the enzymatic inhibitor maintains the structural integrity of the DNA.
S Stabilises your DNA Long Term S Improves DNA Yield and Purity S Room Temperature Storage S Lyses the cells
S Compatible with DDK Isolation Kit
Lanes 1 & 2: freshly taken swabs Lanes 3 & 4: Stored 1 month with Dri-Capsule Lanes 5 & 6: Stored 3 months with Dri-Capsule Lanes 7 & 8: Stored 6 months with Dri-Capsule Lanes 9 & 10: Stored 12 months with Dri-Capsule 0: Negative control
DNA Stability comparison over 5 years using Isohelix DSK Stabilising and Lysis Kit.
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APPENDIX C
INSTRUCTIONS FOR ISOHELIX DNA ISOLATION KITS
ce//xxxxx
projects
Version 6 Updated November 2011
Instructions for Isohelix DNA Isolation kits: DDK-3/DDK-50
Product Details
Isohelix Buccal DNA Isolation Kits have been specifically formulated to produce high DNA yield and purity from buccal swabs. The kits have been fully optimised at Cell Projects for use on buccal cell samples and offer reduced handling times, increased DNA yields and many other important technical benefits for their use in manual, 96-well or other high throughput formats.
Key Benefits
Optimised for buccal cells Protocol integrated to swabs
Fast handling times Manual or high throughput formats
High purity and yield No columns or filtration
No solvent based chemicals Less consumables wastage
Kit Contents
Isohelix DDK DNA Isolation Kits

Catalogue No. DDK-3 DDK-50 Storage temperature
Number of preps 3 50

Solution LS (Lysis buffer) 1.5ml 25ml Room temperature
Solution PK (Proteinase K) 100p! 1 ml -20UC
Solution CT (Capture buffer) 1.5ml 25ml Room temperature
Solution TE (Re-hydration buffer) 500p! 15ml Room temperature
Storage
STORE KITS AT CORRECT TEMPERATURE ON ARRIVAL
Isohelix DNA Isolation Kits are shipped at ambient temperature.
Please note that on arrival the kit components should be stored according to the table above.
The kits are stable up to the expiry date if stored as instructed. See box label for expiry date.
Equipment and reagents to be supplied by user
Water bath or heating block at 60C
Pipettes with disposable tips
Microcentrifuge (with rotor for 2 ml tubes)
1.5ml microcentrifuge tubes
Vortexer
Before Starting
1. Prepare a waterbath at 60C
2. If a precipitate has formed in solution LS, warm at 60C for a few minutes
3. Remove the PK solution from the freezer and allow to thaw at room temperature.
Note: If a fine white powder precipitate has formed in the PK, vortex the tube immediately prior to pipetting the PK. The precipitate should not block the pipette tip and will dissolve when added to the LS buffer.
This precipitate if present has no adverse effect on the activity of the Proteinase K.
Technical Assistance
If you have any questions regarding the use of this kit or other Isohelix products please contact us by email at info@isohelix .com or for further information visit the website at www.isohelix.com
Safety and Use of the DDK DNA Isolation kits
The DDK kits are intended for use by qualified professionals trained in potential laboratory hazards and good laboratory practise. If direct information is not available on any of our compounds this should not be interpreted as an indication of product safety.
This kit has been designed solely for research use only
Isohelix is a division of Cell Projects
For swab or DNA isolation queries email: info@isohelix.com www.isohelix.com
Molecular Biology Solutions www.cellproiects.com Page 1 of 2
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DNA Isolation Protocol
Part A DNA Stabilisation
1. Add 500ul LS solution to the tube containing the buccal swab.
2. Add 20(jl PK solution to the tube containing the buccal swab and LS solution. Vortex briefly.
At this point the DNA is stabilised. You may continue with the DNA isolation or store the stabilised swab in a sealed tube at room temperature for at least 3% years.
Part B DNA Isolation
3. Place the tube containing the swab, LS solution and PK solution in a 60C water bath for 1 hour. Vortex briefly.
4. Re-set the water bath to 80C if you are using step 14.
5. Transfer the liquid in the tube (approx. 400ul) into a 1.5ml centrifuge tube using a sterile pipette tip.
6. Optional step to increase yield:
If using SK-1 swabs, tip the swab head and stick into a sterile 1.5ml centrifuge tube so that the swab head is uppermost. Spin the tube briefly and using a sterile pipette tip add the recovered supernatant to the 400|jl collected previously.
If using SK-2 swabs, spin the SK-2 tube containing the swab head briefly and using a sterile pipette tip add the recovered supernatant to the 400|jl collected previously
7. Add 400pi CT solution to the tube, (500pl if using the optional step 6). Vortex briefly.
8. Place the tube in a microcentrifuge (see tip below) and spin at approx. 13K rpm for 7 minutes to pellet the DNA.
Note the pellet may not be visible.
Tip: Place the tube with hinge positioned outwards so the liquid can be removed from the opposite side.
9. Remove all the supernatant carefully with a pipette tip taking care not to disturb the DNA pellet.
10. Re-spin the tube briefly and remove any remaining liquid.
Note it is important to remove all of the liquid.
11. Add 150pl TE solution to the tube. This volume may be decreased to as little as 30pl if a higher concentration of DNA is required.
12. Leave for at least 5 minutes at room temperature for the DNA to re-hydrate, longer if a reduced volume of TE has been used. Vortex briefly.
13. Optional step (May improve lower than expected A260/280 and A260/230 ratios.) Spin tube at 13K rpm for 2 minutes to remove any undissolved particulates and remove supernatant to a clean tube.
14. Incubate the tube at 80C for 5 minutes. Vortex and spin the tube briefly.
Note do not use this step if you require double stranded DNA for example restriction digests.
The DNA sample is now ready for use in downstream applications such as amplification.
Store the DNA sample at 4C for short term storage or -20C for long term storage.
The expected yield from a buccal swab is 2 to 10pg DNA (10 to 70ng/pl).
Our DQC-50 DNA Quality Check Kit is designed specifically to confirm presence of and to test both the quality and quantity of your human DNA by a quick PCR test before you start downstream testing.
Other Cell Projects Products Isohelix DNA Buccal Swabs.
> High yields, blood alternative, reproducible, easy to use, different formats for various extraction methodologies. Isohelix DNA Silica Gel Capsules
> For use with SK-1 swab kits, air-dries swab in tube giving extended storage times without loss of stability. Isohelix DNA Isolation and Handling kits
> DNA stabilising kits for the stable storage of DNA at Room Temperature for long periods: DSK-3/50
> DNA quality check by PCR to confirm quality of DNA prior onward experimentation: DQC-50
> DNA Release kits quick and easy kit for PCR-ready DNA from buccal swabs in under 20 minutes: BEK-50
> Isohelix Spin+Collect sample recovery devices to increase yields from swabs and other materials: SC/ST-100
PCR Products A range of high quality PCR plastic for 96 well format plates and cap strips
Electroporation The HiMaX electroporation cuvettes and buffers maximise molecular electroporation and electrofusion efficiencies for Bacteria, Yeast, Insect, Plant and Mammalian cells.
2 of 2
Isohelix is a division of Cell Projects
For swab or DNA isolation queries email: info@isohelix.com www.isohelix.com Molecular Biology Solutions www.cellproiects.com


APPENDIX D
EAS DOMAIN AND SUBSCALE DESCRIPTION
Maternal Sensitivity refers to a mothers ability to pick up on infant cues, provide warmth and soothe distress, and to be responsive in a variety of situations through quality affective interactions with her child. An optimally sensitive mother creates a genuine and positive affective environment for the infant, demonstrates a clarity of the infants needs and expressions, and provides appropriate responsiveness to these needs and emotional expressions. Subscales of the Sensitivity scale include: Affect; Clarity of perceptions and appropriate adult responsiveness; awareness of timing; flexibility, variety, and creativity; acceptance; amount of interaction; and conflict situations.
Structuring involves creating learned opportunities through adequate guidance and scaffolding. The mother demonstrates successful attempts of following the infants lead, and sets appropriate limits to discourage maladaptive behaviors. This is done while allowing the child autonomous pursuits and exploration, yet maintaining the older and wiser framework (Bowlby, 1969) in an optimal parent-child relationship. Subscales of the Structuring scale include: provision of guidance; success of attempts; amount of structure; limit setting; remaining firm in the face of pressure; verbal vs. non-verbal structuring; and peer vs. adult role.
Non-Intrusiveness refers to the appropriateness of directedness on the part of the mother, providing balanced stimulation. In an optimal non-intrusive relationship, there is a lack of over-directedness, over-stimulation, interference, and over-protectiveness. The non-intrusiveness scale also holds a high respect for the autonomous pursuits of the child. Overly-involving acts by the mother are alone not be problematic, particularly if warranted by the
73


childs age or developmental abilities, but when they persist in the face of unwelcoming cues by the child, they are considered intrusive. Non-intrusiveness differs from structuring in that the latter is focused on guidance and the former is more about behavioral interference. The subscales of the Non-intrusiveness scale are: Follows childs lead; non-interruptive ports of entry; commands, directives; adult talking; didactic teaching; physical vs. verbal interferences; and made to feel intrusive.
Non-Hostility refers to both overt and covert hostility that can be expressed in language, impatience, or anger. Adult hostility need not solely be directed at the child; rather, any acts of anger, impatience, or boredom in the presence of the child is considered hostile in nature. Consistent with the idea of background anger and the negative influence this can have on child development (Cummings, 1987), any observable signs of hostility are considered when observing the mother-child relationship. The subscales of the Non-Hostility scale include: Adult lacks negativity in face or voice; lack of mocking; lack of threats of separation; does not lose cool; frightening behaviors; silence; and hostile themes of play.
Child Responsiveness refers to the infants reaction to parental bids for engagement, and the expression of pleasure that accompanies this reaction. The focus is on the emotional and social responsiveness toward the mother. Both affect and responsiveness are considered, and a lack of positive emotional expression or ignoring a mothers invitation to engage is considered non-optimal. An optimally responsive child is one who is happy in the presence of the mother and is emotionally and behaviorally responsive to the mother. The Child Responsiveness subscale is closest to the attachment theory view of the secure/insecure child (Biringen, 2008). The subscales of the Infant/Child Responsiveness scales include: Affect/emotional regulation; responsiveness; age-appropriate autonomy seeking; positive
74


physical positioning; lack of avoidance; and task oriented/concentration.
Child Involvement refers to extent to which a child seeks to engage the mother in play as well as other means of actively seeking engagement by the mother. In infants, this may be demonstrated through looking and babbling, and in older children this will include both positive verbal and behavioral means to engage the caregiver. Optimal involvement of the parent by the child lacks negative involving behaviors, and includes appropriate autonomous exploration, engaging the mother at a developmentally appropriate level (Biringen et al., 1998). The subscales of the Child Involvement scale include: Simple initiative; elaborate initiative; use of adult; lack of over-involvement; eye contact; body positioning; and verbal involvement.
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APPENDIX E
COMIRB CERTIFICATE OF APPROVAL
University of Colorado Anschutz Medical Campus
Colorado Multiple Institutional Review Board, CB F490 University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045
303.724.1055 [Phone] 303.724.0990 [Fax] COMIRB Home Page [Web] comirb@ucdenver.edu [E-Mail] FWA00005070 [FWA]
University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center The Children's Hospital University of Colorado Denver Colorado Prevention Center
Certificate of Approval
29~Sep~2014
Ryan Asherin
COMIRB Protocol 14-0851 Initial Application 19-Sep-2014 18-Sep-2015 3
Epigenetic effects of OXTR and 5-HTT on maternal depression and child development
Submission ID: APP001-2 Description:
Response to Deferral
Full Board Initial Application
Investigator:
Sponsor(s):
Subject:
Effective Date: Expiration Date: Expedited Category: Title:
All COMIRB Approved Investigators must comply with the following:
For the duration of your protocol, any change in the experimental design/consent and/or assent form must be approved by the COMIRB before implementation of the changes.
Use only a copy of the COMIRB-approved, stamped Consent and/or Assent Form. The investigator bears the responsibility for obtaining from all subjects "Informed Consent" as approved by the COMIRB. The COMIRB REQUIRES that the subject be given a copy of the consent and/or assent form after it is signed. Consent and/or assent forms must include the name and telephone number of the investigator.
Provide non-English speaking subjects with a certified translation of the approved Consent and/or Assent Form in the subject's first language.
The investigator also bears the responsibility for informing the COMIRB immediately of any Unanticipated Problems that are unexpected and related to the study in accordance with COMIRB Policy and Procedures.
Obtain COMIRB approval for all advertisements, questionnaires and surveys before use.
Federal regulations require a Continuing Review to renew approval of this project within a 12-month period from the last approval date unless otherwise indicated in the review cycle listed below. If you have a restricted/high risk protocol, specific details will be outlined in this letter. Non-compliance with Continuing Review will result in the termination of this study.
You will be sent a Continuing Review reminder 75 days prior to the expiration date. Any questions regarding this COMIRB action can be referred to the Coordinator at 303-724-1055 or UCHSC Box F-490.
Review Comments:
COMIRB noted the Board review and subsequent deferral performed on 30-May-2014. The committee reviewed the Pi's written response to deferral and found it to be satisfactory with the exception of the requested itemized changes below.
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University of Colorado Anschutz Medical Campus
Colorado Multiple Institutional Review Board, CB F490 University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045
303.724.1055 [Phone] 303.724.0990 [Fax]
COMIRB Home Page [Web] comirb@ucdenver.edu [E-Mail] FWA00005070 [FWA]
University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center Children's Hospital Colorado University of Colorado Denver Colorado Prevention Center
Submission ID: CRV001-3
SUBMISSION DESCRIPTION:
Response to Minor Modifications
Your COMIRB Continuing Review submission CRV001-3 has been APPROVED until the expiration date listed above. The investigator will need to submit this research for Continuing Review at least 45 days prior to the expiration date.
Study personnel are approved to conduct the research as described in the documents approved by COMIRB, which are listed below the REVIEW DETAILS section.
Please carefully review the REVIEW DETAILS section because COMIRB may have made red-line changes (i.e. revisions) to the submitted documents prior to approving them. The investigator can submit an amendment to revise the documents if the investigator does not agree with the red-line changes. The REVIEW DETAILS section may also include important information from the reviewer(s) and COMIRB staff.
COMIRB stamps the approved versions of documents in the top right hand corner. Stamped copies of documents are available for download through COMIRB's electronic submission website, eRA(lnfoEd).
Click here for instructions on how to retrieve stamped documents.
REVIEW DETAILS:
Certificate of Approval
16-Sep-2015
Investigator:
Subject:
Review Date: Effective Date: Expiration Date: Sponsor(s): Title:
Epigenetic effects of OXTR and 5-HTT on maternal depression and child development
Ryan Asherin
COMIRB Protocol 14-0851 Continuing Review 9/15/2015
15-Sep-2015
14-Sep-2016
77


University Research
UNIVERSITY OF COLORADO DENVER | ANSCHUTZ MEDICAL CAMPUS
Colorado Multiple Institutional Review Board, CB F490
University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045
303.724.1055 [Phone] 303.724.0990 [Fax] COMIRB Home Page fWebl comirb@ucdenver.edu [E-Mail] FWA00005070 [FWA]
University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center Children's Hospital Colorado University of Colorado Denver Colorado Prevention Center
Certificate of Approval
03-0ct-2016
Investigator:
Subject:
Review Date: Effective Date: Expiration Date: Sponsor(s):
Title:
Expedited Category:
Ryan Asherin
COMIRB Protocol 14-0851 Continuing Review 29-Sep-2016 29-Sep-2016 28-Sep-2017 None~
Epigenetic effects of OXTR, 5-HTT and irregular cortisol reactivity patterns on maternal depression and child development
3,7
Submission ID: CRV002-1
SUBMISSION DESCRIPTION:
Status: Enrolling
Your COMIRB Continuing Review submission CRV002-1 has been APPROVED until the expiration date listed above. The investigator will need to submit this research for Continuing Review at least 45 days prior to the expiration date.
Study personnel are approved to conduct the research as described in the documents approved by COMIRB, which are listed below the REVIEW DETAILS section.
Please carefully review the REVIEW DETAILS section because COMIRB may have made red-line changes (i.e. revisions) to the submitted documents prior to approving them. The investigator can submit an amendment to revise the documents if the investigator does not agree with the red-line changes. The REVIEW DETAILS section may also include important information from the reviewer(s) and COMIRB staff.
78


Full Text

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EFFECTS OF OXTR ON MATERNAL DEPRESSION AND DYADIC EMOTIONAL AVAILABILITY by RYAN M. ASHERIN B.A., Metropolitan State University of Denver, 2004 M.A., University of Colorado Denver, 2014 A dissertation submitted to the Faculty of the Graduate Sc hool of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Clinical Health Psychology Program 201 7

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ii This dissertation for the Doctor of Philosophy degree by Ryan M. Asherin has been appro ved for the Clinical Health Psychology Program by Peter Kaplan, Chair Kevin Everhart Advisor Dave Albeck Jim Grigsby Date: May 13, 2017

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iii Asherin, Ryan M. ( Ph.D., Clinical Health Psychology Program ) Effects of OXTR on Mate rnal Depression and Dyadic Emotional Availability Dissertation directed by Professor Kevin Everhart ABSTRACT Recent research has demonstrated a link between polymorphisms in the oxytocin receptor gene (OXTR ) and increased psychopathology, n on optimal parenting behaviors, and negative outcomes in child development. Genotypic polymorphism of OXTR is expressed through the homozygous or heterozygous pairing of alleles A and G. Comparisons of individuals with different OXTR genotypes have suggest ed that adult carriers of the A allele display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, poor er parenting skills, decreased empathy and worse mental health outcomes compared to homozygous G/G adults (Riem, Pieper, Out, Bakermans Kranenburg, & Van Ijzendoorn, 2011) However, d espite the reported benefits to parenting behavior associated with the homozygous G/G OXTR genotype it has also been associated with a greater risk of developing major depression (R. J. Thompson, Parker, Hallmayer, Waugh, & Gotlib, 2011) In addi tion, given the bidirectional influences of parent and child in both caregiver sensitivity and maternal depression (Feldman, 2012b, 2017) an important unaddressed question concerns effects associated with the child's genotype or the concordance/discordance between dyadic genotyp ic var iations. The objective of this dissertation was to examine relationship s that may exist between mothers and their infants in relation to oxytocin receptor types, maternal depression, and mother and child dyadic emotional availability, as measured by the em pirically supported Emotional Availability Scales ( EAS; Biringen, 2008) F our specific hypotheses were tested : (1) that t he presence of depression in the postpar tum period

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iv will be related to the presence of the G/G OXTR allele sequenc e, (2) that mothers carrying an A OXTR allel e will demonstrate decreased levels of S ensitivity as measured by the EAS (3) that i nfants of depressed mothers who are carriers of an A O XTR allele will be protected from some of the adverse effects of maternal depression and demonstrate reduced dyadic sensitivity, and (4) that n on depressed mother child dyads that are both carriers of the homozygous G genotype will demonstrate significantl y higher dyadic emotional availability ratings. In addition, although specific a priori predictions were not made, analys e s of the unique contributions of infant OXTR genotype and mother infant concordance/discordance on maternal functioning and mother inf ant interactions were carried out in posthoc analysis Mother infant dyads (N= 6 1 ) were recruited using an advertisement posted on Facebook. Mothers were asked to complete demographic questionnaires, several self report depression inventories, a S tructured Clinical Interview for DSM IV Axis I Disorders (SCID) and a 10 minute semi structured play session for later coding using the EAS. Infant cognitive and communicative development were assessed using the Bayley Scales of Infant and Toddler Develop ment (3 rd Edition; BSID II). S alivary sample s were obtained via buccal swab for later genotypic analyses. DNA isolation was preformed according to manufacture specifications (Isohelix DNA Isolation Kits: DDK 3/DDK 50) before genetic sequencing of OXTR rs53 576 was completed through the use of a STEPONE qPCR machine. Contrary to predictions, m aternal OXTR genotype was not associated with increased rates of maternal depression, as measured either by common P P D screening measure s or via structured cli nical interview. Variations in m aternal OXTR genotype alone did not account for a significant difference in global ratings of maternal S ensitivity or C hild R esponsiveness on the EAS nor did it account for child BSID III performance However, both maternal and

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v child OXTR genotype were significantly association with maternal non intrusiveness, with the GG genotype in both cases linked to greater maternal intrusiveness. Furthermore infant OXTR genotype was linked to significant differences in DSM IV Axis I d epression spectrum diagnoses of the mother, as well as to the mother's interviewer rated Global Assessment of Functioning (GAF). W hen infant genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozy gous G genotype, maternal depression diagnosis was significantly more likely to be observed in mothers of GG infants (X 2 (3) =8.440, p =.038). Mothers of GG i nfants had significantly lower GAF ratings than those with at least one A allele ( r =.335, n =58, p =.01 0) Infants displayed more optimally on th e C hild R esponsiveness subscale of R esponsiveness EAS scale when mothers were carriers of an A allele (F(1,59 ) = 3.984, p = .051) Furthermore, on the Affect subscale of the Sensitivity scale, mothers in concordant dyads (i.e. mother/infant GG/GG, AG / AG AA/AA) displayed significantly higher Affect after minority status, and mothers' and infants' genotypes had been separately taken into account F(1,54)=5.685, p=.021, 2=.095) A n effect of genotypic concordance was also obtained on the Interaction subscale of the Sensitivity scale after controlling for minority status and mothers' and infants' genotypes (F(1,54)=5.233, p=.026, 2= .088 ) Similar findings were observed based on genotypic concordance on Non Intrusivene ss subscales of F ollowing C hild L ead (F(1,54)=4.246, p=.044, 2=. 072) and A dult T alking (F(1,54)=5.767, p=.020, 2=. 095). Results of this study suggest that maternal genotype alone may not be enough to discriminate predictors of complex dyadi c interactions. Biobehavioral synchrony between mother and infant and specifically OXTR genotype concordance/discordance, may offer increased insight into the role oxytocin and OXTR play in observable dyadic emotional

PAGE 6

vi availability, as well as the greater development of pair bonding, attachment, and social affiliation. Contrary to expectations from the literature, mothers with the GG OXTR genotype were not more sensitive or more prone to depression. If anything, this genotype was related to greater maternal intrusiveness during a semi structured play interaction. However, GG mothers showed better sensitivity if their infants were GG than AG /AA suggesting either that concordance promotes optimal biobehavioral synchrony and functioning of the oxytocin system or is a proxy for some other, unmeasured, variable that leads to better dyadic interactions. Possible mechanisms to explain the association between infant OXTR genotype and maternal depression, and between OXTR concordance and maternal emotional availabili ty, are discussed. The form and content of this abstract are approved. I recommend its publication. Approved: Kevin Everhart

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vii DEDICATION I dedicate this work to my wife, my son, and my parents Jess: you are my biggest supporter and best fri end. Your love, strength, and support have given me the motivation to continue to pursue my career goals while building a beautiful life together. Thank you for your unconditional love and understanding over the years. My graduate school experience has bee n so much more fulfilling because I have gotten to share it with you. Alfie: I love you more than you'll ever know. You inspire me to be the best version of myself each and every day. Thank you for teaching me what it's all about. Mom and Dad: I have spent a lot of time and energy throughout my academic career trying to gain a better understanding of infant and childhood development. I still have a lot to learn, but I am certain that none of the achievements throughout my life would have been possible witho ut your love, support, and prayers. Thank you for all that you have given me, especially the confidence to believe in myself and never stop pursuing my dreams.

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viii ACKNOWLEDGMENTS I would like to thank the Infant L ab at the University of Colorado Denver, especially my mentors, Drs. Kevin Everhart and Peter Kaplan. I have benefitted from having two primary advisors that are each able to nurture my intellectual growth while offering co nstructive criticism and praise. Within the Infant Lab I would like to acknowledge the support of my fello w graduate student Jo Voge li I appreciate all the time we have spent together pursuing shared interests and ambitions. I would also like to thank Josh Fowler, Shiva Fekri, and Dr Christopher Phiel I am grateful for each of their contributions, which allowed for the successful processing of oxytocin rec eptor genotypes. Josh Fowler, in particular, deserves recognition for his a ssistance, guidance, and friendship while spending long hours in the lab together processing the genotypes that became a central focus of this dissertation. Last, but not least, I would like to thank all the ori ginal members of my PhD cohort, Kellie Martins, Megan Grigsby, Stephanie Hooker, Carissa Kinman, and Jenn Altman Each has been an inspiration to me over the years, and I am truly thankful to be able to call them my friends. In particular, I would like to thank Kellie, Megan, a nd Stephanie for making my time within the program one that has been filled wi th as much laughter as hard work.

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ix T ABLE OF CONTENTS CHAPTER I. BACKGROUND ................................ ................................ ................................ 1 II. METHODS ................................ ................................ ................................ ........ 17 Sample ................................ ................................ ................................ ................ 17 Participants ................................ ................................ ................................ ......... 18 Procedure ................................ ................................ ................................ ........... 21 Measures ................................ ................................ ................................ ............ 24 Data Analysis Plan & Power ................................ ................................ .............. 31 Ethical Considerations ................................ ................................ ....................... 32 III. RESULTS ................................ ................................ ................................ .......... 34 Hypothesis 1: Genotype x Depression ................................ ............................... 34 Hypothesis 2: Genotype x EAS ................................ ................................ ......... 36 Hypothesis 3: Genotype x Infa nt Performance ................................ .................. 37 Hypothesis 4: Concordance x EAS ................................ ................................ .... 40 IV. DISCUSSION ................................ ................................ ................................ .... 43 Null Hypothesis Results ................................ ................................ ..................... 44 Post Hoc Analysis ................................ ................................ .............................. 46 Empirical Significance ................................ ................................ ....................... 49 Study Limitations ................................ ................................ ............................... 51 Conclusion ................................ ................................ ................................ ......... 52 REFERENCES ................................ ................................ ................................ .. 54

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x APPENDIX A. Concordance Poster Presentation ................................ ................................ ....... 67 B. Isohelix Swab Brochure ................................ ................................ ..................... 68 C. Instructions fo r Isohelix DNA Isolation Kits ................................ ..................... 71 D. EAS Domain and Subscale Description ................................ ............................ 73 E. COMIRB Certificate of Approval ................................ ................................ ..... 76

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xi LIST OF TABLES Table 1. Demographic data by genotype ................................ ................................ ................................ ............ 20 2. Emotional Availability Scales and Subscale Descriptions ................................ ................................ ... 29 3. Depression results by mother and infant genotype ................................ ................................ ............... 35 4. EAS correlations of direct scores ................................ ................................ ................................ ......... 36 5. Non Intrusiveness direct score MANCOVA result ................................ ................................ .............. 37

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xii LIST OF FIGURES Figure 1. Genetic variants in the oxytocin receptor gene (OXTR) ................................ ................................ ........ 5 2. Biobehavioral synchrony in human attachments ................................ ................................ .................. 10 3. Power based on sample size for a 3x3 ANOVA ................................ ................................ .................. 32 4. BSID III percentile rank by ge notype ................................ ................................ ................................ .. 39 5. Concordance by significant EAS subscale ................................ ................................ ........................... 42

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xiii LIST OF ABBREVIATIONS OXT Oxytocin OXTR Oxytocin Receptor CNS Ce ntral Nervous System PNS Peripheral Nervous S ystem PVN P araventricular Nuclei SON S uraoptic Nuclei BDI II Beck Depression Inventory, 2 nd Edition EPDS Edinburgh Postnatal Depression Scale PDSS Postpartum Depression Screening Scale SCID Structured Cli nical Interview for DSM IV TR BSID III Bayley Scales of Infant Development, 3rd Edition EAS Emotional Availability Scales LS L ysis PK P roteinase K Cog Cognitive Scale RC Receptive Communication Scale EC Expressive Communication Scale SE Social Emotional Scale COMIRB Colorado Multiple Institutional Review Board

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1 CHAPTER I BACKGROUND The evolutionary roots of oxytocin (OXT) date back over seven hundred million years, a s its important role in reproduction has existed, with few modifications, for both invertebrates and vertebrates (Donaldson & Young, 2008; Gimpl & Fahrenholz, 2001) In fact, the word oxytocin was derived from the Greek word kytokÂ’n # meaning "quick birth ," coined by the British pharmacologist Sir Henry Hallet Dale in 1906 after he discovered OXT 's role in stimulating uterine contractions (Dale, 1906) Soon after, two separate laboratories reported OXT 's role in lactation (Ott & Scott, 1910; Schafer & Mackenzie, 1911) Several decades later, laboratory studies performed by Vincent du Vigeaud at Cornell University allowed OXT to become the first human polypeptide hormone sequenced and biochemically synthesized (Vigneaud, Ressler, Swan, et al., 1953; Vigneaud, Ressler, Swan, Roberts, & Katsoyannis, 1954; Vigneaud, Ressler, & Trippett, 1953) Due to this work, OXT is now under stood to be a nonapeptide, or a single linear chain consisting of nine amino acids (Cys Tyr Ile Gln Asn Cys Pro Leu GlyNH2) with a sulfur bridge between the two cysteines amino acids (National Center for Biotechnology Information, 2012a) Largely due to research involving animals models, and particularly rodents (Ludwig & Leng, 2006) OXT has been found to serve dual roles as both a neurotransmitter/neuromodulator in the central nervous system (CNS) and a hormone in the peripheral nervous system (PNS ; Macdonald & Macdonald, 2010) Spe cialized cells in the paraventricular (PVN) and suraoptic (SON) nuclei of the hypothalamus produce OXT (Macdonald & Macdonald, 2010) which is then stored in the axon ter minals of the posterior pituitary gland (Ross et al., 2009) Within the CNS, OXT exerts its central effects th rough

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2 axonal connections from the parvocellular neurons in the PVN to critical brain regions and through volume effects in areas with oxytocin receptors (Macdonald & Macd onald, 2010) In this way, oxytocin's effects rely at least in part, on both dynamic (i.e. hormone driven) and static (i.e. genetically determined) changes to the location and density of receptors within the brain (Macdonald & Macdonald, 2010). Among the areas of the brain targeted by the release of OXT is the amygdala which has a high concentration of OT receptors and whose functioning is associated with OXT levels (Gimpl & Fahrenh h olz, 2001; Macdonald & Macdonald, 2010) The release of the hormone and neurotransmitter OXT throughout the body and brain (Gimpl & Fahrenholz, 2001; Inoue et a l., 1994) is believed to influence a wide range of socioemotional functioning and social behavior (Campbell, 2010; Heinrichs, Von Dawans, & Domes, 2009; Ishak, Kahloon, & Fakhry, 2011; Kemp & Guastella, 2011; UvŠnas Moberg, Arn, & Magnusson, 2005) OXT's role in socioemiotonal functioning and social behavior has been studied, and continues to be studied, extensively in animal models, and particularly in rodents (Insel, 2010) In female rats, maternal behavior has been shown to be regulated, at least in part, by OXT, and affiliative behavior in prairie voles has also been found to be mediated by the nona peptide (Gimpl & Fahrenholz, 2001; Insel, 2010) Additionally, OXT has been associated with increased prosocial caregiving behaviors in humans Given the importance of peripheral OXT production for childbirth and lac tation, it is not surprising that prosocial parenting behaviors, especially those related to infant attachment and pair bonding, seem to correlate with OXT levels. From an evolutionary perspective, OXT plays a vital function in the production of offspring and maintenance of the offspring 's health. S everal studies have successfully demonstrated that the prosocial behavior of caregivers, especially practices of

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3 breast feeding, skin to skin contact ("tender touch"), and use of infant directed speech, are linke d to circulating OXT levels (Barrett & Fleming, 2011; Feldman, Gordon, & Zagoory Sharon, 2010; Feldman, Weller, Zagoory Sharon, & Levine, 2007; Nelson & Panksepp, 1998; Swain, Lorberbaum, Kose, & Strathearn, 2007) Feldman et al., (2010) demonstrated that tender touch between mothers and infants, compared to only minimal contact, naturally increased salivary OXT levels between pre and post interaction saliva collections. Feldman et al., reported a similar finding for fathers in r espect to their own OXT levels, after short term stimulatory contact, compared to fathers that only engaged in mi nimal touch with their infants. Moreover, no significant difference was found between the maternal or paternal OXT levels observed (Feldman et al., 2010) Although OXT has been found to have important implicat ions for both animals and humans behaviorally OXT is currently only known to have one rec eptor (OXTR) that is present in both the CNS and PNS (Gimpl & Fahrenholz, 2001; Lee, Ma cbeth, Pagani, & Young, 2009) The OXTR gene, localized on chromosome 3p253.3, has been found to encode the oxytocin receptor (National Center for Biotechnology Information, 2012b; Simmons, Clancy, Quan, & Knoll, 1995) which is known to be a G protein coupled receptor that binds to the hormone/ neuropeptide OXT and triggers a structural change to the receptor promoting the activation of downstream G proteins and the subsequent rele ase of calcium ions from intercellular stores (Gimpl & Fahrenholz, 2001; Zingg & Laporte, 2003) Since OXT released from the pituitary gland is not able to re enter the central ner vous system due to the blood brain barrier, it is believed that the behavioral effects of OXT are produced by the centrally projecting oxytocin receptors and expressed by neurons located throughout the brain and spinal cord (Ross et al., 2009) However, the specific effects of OXTR's activation

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4 appear to vary by tissue, organ, and species (Zingg & Laporte, 2003) and the specific distribution of OXTR in the brain has been difficult to model as positron emission tomography radioligand have not yet been developed with appropriate CNS penetratio n or receptor specificity to accurately model although some work in animal models continue to make progress in this area of research (Baribeau & Anagnostou, 2015) Furthermore, the study of OXTR is complex as it has been found to have at least 28 unique single nucle otide polymorphisms (SNPs) across four exons and three introns (National Center for Biotechnology Information, 2012 a) Recent research has focused on two particular SNPs (i.e. rs53576 and rs2254298) in the third intron (Figure 1) that have been correlated with social affiliation, parenting, empathy, response to stress, and psychopathology (Bakermans Kranenburg & van Ijzendoorn, 2014) In particular, SNP rs53576 has received growing attention for its connection to prosocial parenting behaviors (Bakermans Kranenburg & Van Ijzendoorn, 2008; Klahr, Klump, & Burt, 2015) while rs2 254298 is often studied in relation to symptoms of autism spectrum disorder (Francis et al., 2016) With this in mind, OXTR SN P rs53576 was selected for study in relation to mother infant dyads.

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5 Figure 1 Genetic variants in the oxytocin receptor gene (OXTR) Note: The (OXTR) gene is located on chromosome 3p25 3p26.2, spans 17 kb, and contains three intro ns and four exons. Th is 389 amino acid polypeptide with seven transmembrane domains belongs to the class I G protein coupled receptor. Two single nucleotide polymorphisms (SNPs) in the third intron of OXTR have emerged as particularly promising candidates in the study of sociobehavioral phenotypes (indicated in bold): rs53576 (G/A) and rs2254298 (G/A). The main SNPs with their location and rs number are shown ab ove and rs53576, the focus of this study, is denoted with a star Exons are indicated by the dar ker shaded boxes, and the untranslated regions are shown by the lighter boxes. Variants in the gene are shown by arrows. Chr, chromosome (Kumsta & Hei nrichs 2013) Genotypic polymorphism of OXTR SNP rs53576 is expressed through the homozygous or heterozygous pairing of alleles A and G (Rodrigues, Saslow, Garcia, John, & Keltner, 2009) Although the underlying processes linking variants of the OXTR gene to actual OXT levels in humans have not yet been clarified, at least one study has sug gested that OXTR rs53576 may contribute to oxytocin's ability to bind with its receptor (Tost et al., 2010) Despite the uncertainty regarding the underlying mechanism that may relate OXTR to socioemotional functioning and social behavior, c omparison of OXTR genotypes have suggested that carriers of an A allele (i.e. G/A and A/A) display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, relatively poor parenting skills, decrea sed empathy and deleterious mental health outcomes compared to individuals

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6 with a homozygous G/G allele pair (Bakermans Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010) Interestingly though, depressive symptomatology may suppress the positive effects of the homozygous G/G allele pair (Riem et al., 2011) Despite the reported benefits of parenting behaviors associated with the homozygous G/G OXTR allele pair, it has also been associated with a greater risk of developing major depression (R. J Thompson et al., 2011) A 2013 study (N=288) reported that carriers of a G allele, particularly in the presence of early life adversity, were associated with increased vulnerability to depression symptoms (McQuaid, McInnis, Stead, Matheson, & Anisman, 2013) which contrasted the results of an early study (2011) that linked depression symptomatology to carriers of an A allele (Saphire bernstein, Way, Kim, Sherman, & Taylor, 2011) However a longitudinal study (N=155) conducted by Feldman et al. found that not onl y did depressed mothers show decreased levels of OXT but chronically depressed mother child dyads were four times more likely to have a homozygous G/G OXTR allele pair than non depressed mothers (Feldman, 2012b) Although individual studies have supported the idea of O XTR being associated with socia bi lity, parenting behaviors, and psychopathology, it remains unclea r how generalizable these findings are despite their reported statistical significance. Like many genetic association studies, results can often be difficult to replicate (Ioannidis, Ntzani, Trikalinos, & Contopoulos Ioannidis, 2001) As the published literature regarding OXTR rs 53576 has grown, meta anal ytic studies have been completed on OXTR's purported role in sociability and psychopathology. A 2014 meta analysis included the reported effect sizes for 52 samples (N = 17,557 participants) that analyzed OXTR rs53576 in relation to one or more of five

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7 dis tinct domains (i.e. biology, personality, social behavior, psychopathology, and autism) as categorized by the authors (Bakermans Kranenburg & van Ijzendoorn, 2014) Based on their systematic review of the OXTR rs53756 literature and subsequent meta analysis, none of the five domains demonstrated a significant main effect with OXTR which led the stud y's authors to conclude that OXTR SNP rs53576 does not significantly explain the complexities of human social behavior However, an even more recent systematic review and corresponding meta analysis challenged these resu lts, particularly as OXTR rs5357 6 relates to sociability (Li et al., 2015) Li et al. (2015) deconstructed the social beha vior domain used in Bakermans Kranenburg & van Ijzendoorn's (2014) study and recategorized phenotypic outcomes reported in all r elevant published research into a general sociality domain (e.g. extraversion, empathy, and social lon e liness) or a sociality w ithin the context of close relationships domain (e.g. maternal sensitivity, child/adult attachment, and marital quality) as well as teasing apart reported effects on depression from a larger psychopathology domain. Utilizing data available from eighteen s tudies and twenty four unique samples (N=4,995) that reported phenotypic outcomes related to general sociality, the autho rs found that homozygous GG allele OXTR genotypes demonstrated increased social attributes compared to carriers of an A allele. However a significant result was not found when a separate analysis of ten studies and fifteen inde pen dent samples (N=5,262) was conducted to look at the association between OXTR rs53576 and quality of close relationships. Collectively, these meta analytic resul ts led Li et al. (2015) to posit that OXTR rs53576 polymorphism predicts how an individual will generally relate to others, but not within the unique context of close relationships.

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8 Moreover, both Bakermans Kranenburg & van Ijzendoorn (2014) and Li et al. 's (2015) meta analyses failed to show an association between OXTR and psychopathology specifically, depression. However, it should be noted that the samples included within their respective meta analyses were quite heterogeneous including samples from d ifferent countries, both sexes, and diverse psychiatric populations. Biological and epidemiological evidence suggests that PPD is unique to other forms of depression with regard to biopsychosocial risk factors (Kimmel et al., 2016) and previous research has demonstrated that OXTR's genotype is expressed phenotypically different ly dependent on cultural norms (Feldman, Monakhov, Pratt, & Ebstein, 2016; H S Kim et al., 2011) N o study has specifically reported on the association between OXTR rs53576 and PPD status although a recent study demonstrated an interaction between perinatal OXTR methylation and development of PPD (Bell et al., 2015; Serati, Redaelli, Buoli, & Altamura, 2016) Therefore it is worth continuing to investigate associations between OXTR and PPD, despit e the null findings of recent meta analytic studies regarding OXTR and depression phenotype. Although meta analytic studies have drawn concern for OXTR's purported role in sociability and psychopathology, and even the quality of close relationships, publis hed studies continue to report associations between OXTR rs53576 and parenting (Bakermans Kranenburg & Van Ijzendoorn, 2008; Klahr et al., 2015; Marsh et al., 2012; Michalska et al., 2014) In Li et al.'s (2015) meta analysis, which did not find a significant association between OXTR and sociality within the con text of close relationships, we again see a heterogeneous mixture of diverse adult focused samples included in their analysis of OXTR in relation to close relationships. Phenotypic outcome measures of the included samples ranged from pair bonding (Walum et al., 2012) separatio n anxiety (Costa et al., 2009) and

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9 attachment security/anxiety (Chen & Johnson, 2012; Costa et al., 2009; Gillath, Shaver, Baek, & Chun, 2008; Krueger et al., 2012; Luijk et al., 2011; Rodrigues et al., 2009) to marital discord (Bakermans Kranenburg & Van Ijzendoorn, 2008) interpartner conflict (Sturge Apple, Cicchetti, Davies, & Suor, 2012) and romantic relationship security (Raby, Cicchetti, Carlson, Egeland, & Collins, 2013) Although both recent meta analys es included reported effect sizes from a multitude of published studies that demonstrated p hen otypic outcomes related to parent child re lationships, parenting behavior was not the specific focus when considering sociability or even sociability within the c ontext of close relationships. Perhaps just as Li et al. (2015) proposed an inherent difference between general sociability and sociability within the context of a close relationship, there may be a significant difference in the functioning of the oxytocin ergic system between mother infant dyads. Recent work has led to an appreciation for a complex synchronous relationship that occurs between an individual's environment and biological factors that can influence both the mother and infant in the perinatal a nd postpartum sensitive period s particularly in relation to bond formations, parenting behaviors, and attachment (Feldman, 2016) Insight from early rodent and animal models into the association between pair bonding and the oxytocin system has continued with the study of humans (Carter, 2014) A leading conceptualization of the role oxytocin plays on human attachment formation stresses the importance of biobehavioral synchrony or a coordinated biological and behavioral process that occurs d uring social interactions between mother infant dyads ( F igure 2; Feldman, 2017) This conceptualization is noteworthy within the study of mother infant dyads, as the oxytocinergic system is being shaped in infancy. In fact, reports have demonstrated that behavioral interactions between mother infant dyads in early lif e are partially responsible for the

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10 orga n ization of oxytocin avail a bility and OXTR localization in the brain (Dumas, Nadel, Soussignan, Martineri e, & Garnero, 2010; Weaver et al., 2004) From an epigenetic framework, this early life experience and the corresponding oxytocinergic system development shapes the ability for an individual to go on to form attachments and ultimately parent the next gene ration ( for a review of the neurobiology of human attachments, please see Feldman, 2017) Figure 2 Biobehavioral synchrony in human attachments Note: Human attachments are characterized by the coupling of the partners' physiological an d behavioral processes during moments of social contact. Such coupling is observed across four systems: matching of nonverbal behavior, coupling of heart rhythms and autonomic functioning, coordination of hormonal release, and brain to brain synchrony Abb reviations: HR, heart rate; OT, oxytocin (Feldman, 2017) Several previously published studies have specificall y reported on associations between OXTR rs53576 genotype and maternal behavior. In a pioneering study published in 2008, Bakermans Kranenburg & van Ijzendoorn became the first to report a relationship between mothers (N= 159) carrying a homozygous G allele genotype and increased sensitive parenting. A study then came out linking non mothers with low depression scores (N=40) to increased heart rate responses to infant cries when carriers had the GG genotype (Riem et al.,

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11 2011) Marsh et al. (2012) followed with a significant f inding associating mothers (N=57 ) with increased infant face preference following intranasal OXT administration in carriers of the homozygous G allele sequence. A somewhat more complex study (N=40) followed that retrospectively associated maternal carriers of the A allele with increased levels of prosoci al parenting (observed when the child was 4 6 years of age) and neural activation of prosocial parenting regions of the brain (i.e. bilateral orbitofrontal cortex and left anterior cingulate cortex) when viewing photographs of their children fifteen years later (Michalska et al., 2014) Although Michalska et al. 's (2014) finding contradicts prior studies demonstrating a link between the heterozygous G OXTR rs53576 genotype and prosocial parenting behaviors it did still report a significant finding betwee n OXTR polymorphism and parenting. Perhaps most recently, Klahr et al. (2015) reported that in a sample of five hundred twin families (N=1000 twins, n=494 mothers), maternal carriers of the homozygous G allele predicted greater warmth with her offspring, e ven after controlling for the child's genotype. This latter study was nove l, in that it considered both direct (i.e. maternal genotype) and indirect (i.e. child genotype) genetic influences (Klahr et al., 2015) The indirect genetic influences of the child were considered due to meta analytic findings highlighting the importance of not only parental, but also child, genetic factors on parenting behavi ors due to an "evocative gene environment correlation" (Klahr & Burt, 2014 p.547 ) Although these findings are quite notable and speak to the potential importance of considering dyadic gene x environment research designs Klahr et al. (2015) utilized a complex measurement system involving confirmatory factor analysis based on two objective parent/child coding systems (i. e. joystick coding method and the more global Parent Child Interaction System coding method) for obtaining parent composite scores statistically

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12 associated with warmth, control, and negativity dimensions that mapped onto prior behavioral genetic studies of parenting originating from their workgroup (Klahr & Burt, 2014) However, few studies have specifically addressed the relationship between genotypic and phenotypic expre ssions of OXTR rs53576 on maternal depression and in relation to dyadic emotional availability. The construct of emotional availability' was originally used to describe the unique interaction between a mother and child, while emphasizing the role of emoti onal expression on dyadic interactions (Emde, 2012) Emotional availability's early conceptualization incorporated emotional expression and responsivity into work regarding attachment theory (Bowlby, 1953, 1969, 1973, 1979, 1980) Since then the construct of emotional availability has been reconceptualized based on research regarding the quality of parent child interactions (Biringen, 2000) and is now used in the research setting to describe dyadic interactions across s everal parental dimensions ( S ensitivity, S tructuring, N on I ntrusiveness, N on H ostility) and two child dimensions ( R esponsiveness to parent al bids for attention and I nvolvement of the parent in play ). Given the above overview of empirical support for a pote ntial relationship between OXT and OXTR on prosocial parenting behaviors and sensitivity, it is believed that OXT and OXTR may influence observed emotional availability. For instance a recent study conducted in the Netherlands demonstrated a causal linkag e between intranasal OXT administration and paternal emotional availability (Naber, van Ijzendoorn, Deschamps, van Engeland, & Bakermans Kranenburg, 2010) and, most notably, Bakermans Kranenburg & van Ijzendoorn's (2008) pioneering study l ooking at prosocial parenting behaviors demonstrated that mothers homozygous for the G allele pair of OXTR SNP rs53576 exhibited increased sensitivity

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13 The focus of this study was to examine the synchronous relationship that may exist between mothers, with or without depression, and their infants. The polymorphic arrangement of OXTR rs53576 allele pairings may be associated with an increased risk of poor infant development if confronted with environmental adversity (maternal depression) as supported by pri or studies Meaney (2001) illustrated a theoretical model for the cross generational transmission of OXTR genotypic expression related to impoverished maternal care. Their model was largely based on empirical reports investigating the effects of environmen tal stress on maternal care in bonnet macaque populations and rodents (Coplan et al., 1996, 1998; Meaney, 2001; Rosenblum & Andrews, 1994) however more recent findings in human mother infant dyads suffering from postpartum depression may offer additional support for Meaney's model In one study r oughly 60% of the children raised by depressed mothers would develop a psychiatric disorder within the first six years of life (Feldman, 2012b) but children of depressed mothers that carried at least one copy of an A OXTR allele had increased OXT levels, significantly reduced psychopathology and expressed empathy that was comparable to a control population (Feldman, 2012b) This finding in part, led Feldman to posit "that in the context of risk associated with maternal infant bonding, more optimal functioning of the [oxytocin] system may promote resilience and provide a buffer that mitigates some of the effects of maternal depression on children's soc ial competence, empathy, and proneness to psychopathology" (Feldm an, 2012b p. 387 ) However, this finding is contrasted by a study that reported that children (N=441) inheriting an A OXTR allele, while raised under the presence of maternal depression (n=41) are more susceptible to developing depression themselves in their adolescence (Thompson, Hammen, Starr, & Najman, 2014) By analyzing maternal depression in relation to the emotional availability of

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14 mother child dyads we can attempt to distinguish how OXTR polymorphic variations on SNP rs53576 influences dyadic emotional availability development under adverse environment al conditions (postpartum depression) which may serve as an important antecedent condition for the contras ting reports on the association between OXTR and the intergenerational transmission of psychopathology. Hypothesis and specific aims This study wa s designed to determine the genotype of OXTR rs53576 for a population of mother child dyads. The study 's aim was to correlate specific OXTR genotypes with maternal depression in the postpartum period, dyadic emotional availability ratings, and infant development. Within this objective, four specific hypotheses were investigated: 1. Genotype x Depression Despite the finding of the more recent meta analysis suggesting that there is not an association between OXTR rs53576 and depression (Li et al., 2015) this study when originally designed predicted that t he presence of depression in the postpartum period will be related to the presenc e of the G/G OXTR allele sequence in the mother similar to Thompson et al 's (2011) and McQuaid et al.'s (201 3 ) finding s linking depression symptomatology with OXTR genotype 2. Genotype x EAS Given the published literature suggesting that carriers of an O XTR rs53576 A allele (i.e. A/G and A/A) display a heightened sensitivity to stress, impaired social skills, decreased reactivity to infant crying, relatively poor parenting skills, and decreased empathy compared to individuals with a homozygous G/G allele pair (Bakermans Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Ki m et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010) this study predicted that m others

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15 carrying an A OXTR genotype will demonstrate decreased levels of S ensitivity as measured by the E motio nal A vailability S cales 3. Genotype x Infant Performance Recent literature studying mother child dyads in relation to OXTR have suggested that there may be a "protective factor" associated with the genotype of the child (Feldman et al., 2010; S. M. Thompson et al., 2014) Although published findings appear to contrast as to whether an A or a G allele predicts more optimal socioemotional functioning of the child this study attempted to operationalize the "protective fac tor" outcome by analyzing infant genotype in relation to two performance based assessments (i.e. Child R esponsiveness direct score on the EAS and Cognitive Development score on the Bayley's Scales of Infant Development ) collected as part of this study. As such, this study predicted that i nfants of depressed mothers who are carriers of an A OXTR allele inherit a protective genetic factor leading to: a. increased ratings of C hild R esponsiveness on the E motional A vailability S cales. b. i ncreased C ognitive D evelopme nt scores, as measured by the Bayley's Scales of Infant Development, 3 rd Edition, relative to those without an A allele. 4. Concordance x EAS A c onceptualization of the oxyto ci nergic system in dyads relying on biobehavioral synchrony for optimal functioning (Feldman, 2012a) coupled with literature suggesting that homozygous G carriers demonstrate more optimal pr osocial parenting behaviors (Bakermans Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klahr et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al.,

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16 2010) led to the original hypothesis that n on depressed mother child dyads that are both carriers of the homozygous G g enotype will demonstrate significantly higher dyadic emotional availability ratings than dyads that did not meet this criterion Interestingly, though, since the s e original hypotheses were made Klahr et al. ( 2014, 2015) has offered compelling evidence th at supports a conceptual biobehavioral synchrony model (Feldman, 2012a) for considering evocative gene x e nvironment interactions in gene association studies. Moreover, studies associating child OXTR genotype to positive outcomes (Feldman et al., 2010; S. M. Thompson et al., 2014) makes one inquire about the bidirectional effect that child genotype might have on parental socioemotional functioning or vice versa At around the same time Klahr et al. and Thompson et al. published their studies our own lab utilized pilot data from this study in preparation of a poster presentation submission (Appendix A ) that showed emerging evidence for an effect on the concordance, or sameness, between mother child OXTR genotypes with maternal depression and dyadic emotional availability (Asherin et al., 2016) To further investigate the possible role that genotype synchrony may have on assessments of maternal depression and dyadic emotion al availability, posthoc analysis was conducted that considered the infant genotype in relation to maternal depression maternal and infant genotype e ffects on dyadic emotional availability separately, as well as categorically defined based on dyadic genot ype concordance (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant) Posthoc analysis results are included within the hypothe s is that was being tested that directly related to the measures of depression, emotional availability, and infant dev elopment.

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17 CHAPTER II METHODS Sample A prospective observational case control design was utilized for this study to draw inferences about an antecedent condition (OXTR genotype) that may be associated with levels of a dependent variable ( maternal psycho pathology dyadic emotional availability and infant development) (Kazdin, 2003) Cases were cons i dered participants with a current or past major depressive episode, as this diagnostic information was collected along with the additional study relevant dependent variables as part of a larger study aimed to assess infant's attention to infant directed speech and cognitive development based on the depressive status of caregi vers (Kaplan et al., 2014; Kaplan, Danko, Diaz, & Kalinka, 2011; Kaplan, Danko, Kalinka, & Cejka, 2012; Kaplan, Sliter, & Burgess, 2007) Data providing the basis for classifying participants with respect to the antecedent condition (OXTR genotype) were collected after participants signed an additional consent form for the collection of salivary samples from both mothers and infants. All research related methods and procedures were approved by the local university affiliated institutional review board (COMIRB protocol #1 1 1641 & COMIRB protocol #14 0851 ). Mothers with and without a history of depression were included in the study. Inclusion criteria was infants between 3 24 months with mothers that are 18 years of age or older Participants were screened and excluded pr ior to enrollment, if the infant ha d a history of serious medical complications but no participants had to be excluded on this criterion A total of sixty two mother infant dyads consented to have their salivary samples collected but one dyad was not abl e to provide a sample due to study personnel error in not collecting the sample prior to them exiting the study Of the

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18 remaining sixty one dyads, genotypic information was collected and OXTR genotype was identified for all but one infant. Once in the stud y protocol the mother and infant dictate the study's progression breaks, diaper changes, feedings, fussiness, or need for naps caused some participants to exit the study prior to all data being collected. However, d ep endent variables were collected for the major ity of dyads with all dyads (N=61) providing demographic information, 60/61 mothers completing a depression screener, 59/61 mothers undergoing a structured clinical interview, 60/61 completing the dyadic emotional availability task, and 57/61 infa nts completing the measure of cognitive development. All available data for the sixty one mother infant dyads was included in the resulting analyses. Participants Table 1 presents demographic information for the full sample of mothers and infants (N = 61) that had salivary samples collected as part of this study All participating mothers (n (mom) =61) had OXTR rs53576 successfully genotyped by utilizing the salivary collection, DNA isolation, and genotyping procedure detailed below. One infant's genotype was considered unidentifiable after two attempts at genotyping, and, thus, subsequently excluded from final analyses (n (infant) =60). No significant differences were found as a function of maternal genotype (i.e. GG, GA, or AA) on infant gender, maternal age, infant age, ethnicity, marital status, maternal level of education, income, number of offspring, depression screening score, depression diagnosis, or G lobal A ssessment of F unctioning (GAF) scores as recorded during the structured clinical interview. When carriers of an A allele (i.e. GA/AA) were collapsed into a distinct group, minority status was found to be significantly higher compared to those with a homozygous G allele pair (t (58) = 2.544; p=0.014)

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19 No significant differences were found as a function of infant genotype (i.e. GG, GA, or AA) on infant gender, maternal age, infant age, ethnicity, marital status, income, number of offspring, or depression diagnosis When carriers of an A allele (i.e. GA/AA) were collapsed into a distinct group, maternal ed ucation level was found to be significantly higher compared to those with a homozygous G allele pair ( t (58) = 2. 332 ; p=0. 0 23 ) Additionally, infant genotype was found to be significant in relation to measures of depression and GAF as discussed under the r es ults for H ypothesis 1 : Genotype x Depression No significant differences were found as a function of dyadic genotype concordance on infant gender, maternal age, infant age, ethnicity, marital status, income, number of offspring, depression screening score, depression diagnosis, or GAF scores However maternal education level was found to be significantly higher in discordant dyads compared to those categorized as concordant (t (58) = 2. 271 ; p=0.0 27) Additionally, t he null hypothesis of Hardy Weinberg equili brium cannot be rejected for either maternal (x 2 =1.53 ; p=0.22 ) or infant (x 2 = 0.12 ; p=0.73 ) genotype (Rodriguez, Gaunt, & Day, 2009) suggesting that the study sample did not significantly deviate from an expected genetic equilibrium inherent in the population and that results were unlikely to be due to errors in genotyping (Hosking et al., 2004)

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20 Table 1 Demographic data by genotyp e Variable Maternal Genotype Infant Genotype Concordant N = 61 dyads GG GA AA GG GA/AA GG GA AA GG GA/AA Yes No n (mom) = 61 33 21 7 33 28 33 21 7 33 28 37 23 n (infant) = 60 31 25 4 31 29 31 25 4 31 29 37 23 Infant Gender (f/m) 16/15 12/13 2/2 16/15 14/ 15 16/15 12/13 2/2 16/15 14/15 21/16 9/14 Age of mother (years) 27.8 (4.6) 28.1 (5.0) 30.4 (3.4) 27.8 (4.6) 28.71 (4.7) 27.8 (4.7) 28.7 (4.8) 28.75 (1.7) 28.7 (4.5) 27.8 (4.7) 28.1 (5.0) 28.6 (3.9) Age of Infant (days) 211 (83.6) 241 (91.9) 270 (90.0) 21 1 (83.6) 245 (90.6) 211 (83.6) 241 (91.9) 270 (90.0) 211 (83.6) 245 (90.6) 219.8 (90.3) 240.4 (84.4) Ethnicity White 29 (87.9%) 13(61.9%) 4 (57.1%) 29 (87.9%) 17 (60.7%) 26 (83.9%) 18 (72.0%) 2 (50.0%) 26 (83.9%) 20 (69.0%) 31 (83.8%) 15 (65. 2%) Minority 4 (12.1%) 8 (38.1%) 3 (42.9%) 4 (12.1%) 11 (39.3%) 5 (16.1%) 7 (28.0%) 2 (50.0%) 5 (16.1%) 9 (31.0%) 6 (16.2%) 8 (34.8%) African American 0 (0.0%) 2 (9.5%) 1 (14.3%) 0 (0.0%) 3 (10.7%) 1 (3.2%) 1 (4.0%) 0 (0.0%) 1 (3.2%) 1 (3.4%) 0 (0/0%) 2 (8.7%) Asian 1 (3.0%) 2 (9.5%) 0 (0.0%) 1 (3.0%) 2 (7.1%) 1 (3.2%) 2 (8.0%) 0 (0.0%) 1 (3.2%) 2 (7.0%) 1 (2.7%) 2 (8.7%) Latina 1 (3.0%) 4 (19.0%) 0 (0.0%) 1 (3.0%) 4 (14.3%) 2 (6.5%) 2 (8.0%) 1 (25.0%) 2 (6.5%) 3 (10.3%) 3 (8.1%) 2 (8.7%) Native Ameri can 2 (6.1%) 0 (0.0%) 2 (28.6%) 2 (6.1%) 2 (7.1%) 1 (3.2%) 2 (8.0%) 1 (25.0%) 1 (3.2%) 3 (10.3%) 2 (5.4%) 2 (8.7%) Marital Status Married 22 (66.7%) 12 (57.1%) 6 (85.7%) 22 (66.7%) 18 (64.3%) 23 (74.2%) 13 (52.0%) 4 (100%) 23 (74.2%) 17 (58.6 %) 23 (62.2%) 17 (74.0%) Unmarried 11 (33.3%) 8 (38.1%) 1 (14.3%) 11 (33.3%) 9 (32.1%) 8 (25.8%) 11 (44.0%) 0 (0.0%) 8 (25.8%) 11 (38.0%) 13 (35.1%) 6 (26.0%) Mother's Education 5.2 (1.2) 5.4 (1.1) 6.1 (0.7) 5.2 (1.2) 5.6 (1.1) 5.1 (1.0) 5.8 (1.1) 5.8 (1 .3) 5.1 (1.1) 5.8 (1.1) ** 5.2 (1.2) 5.8 (0.9) ** Family Income 6.4 (2.1) 6.2 (2.6) 6.7 (2.2) 6.4 (2.1) 6.4 (2.5) 6.1 (2.2) 6.8 (2.3) 6.8 (2.5) 6.1 (2.2) 6.8 (2.3) 6.1 (2.4) 7.0 (1.9) Number of children 1.5 (0.6) 1.4 (0.5) 1.6 (0.8) 1.5 (0.6) 1.4 (0.6) 1.5 (0.6) 1.5 (0.7) 1.0 (0.0) 1.5 (0.6) 1.4 (0.6) 1.4 (0.5) 1.7 (0.6) Depression Screen BDI II score 12.3 (9.8) 10.2 (6.8) 8.4 (3.8) 12.3 (9.8) 9.7 (6.1) 13.2 (10.2) 9.2 (5.6) 7.7 (3.1) 13.2 (10.2) 9.0 (5.3) 12.5 (9.0) 9.2 (7.2) PDSS score 63.1 (21.5) 68.3 (25.0) 51.9 (13.3) 63.1 (21.5) 64.0 (22.2) 67.3 (26.5) 58.7 (16.6) 65.3 (13.1) 67.3 (26.5) 59.6 (16.1) 66.2 (21.4) 59.7 (23.8) EPDS score 6.8 (5.6) 6.1 (4.1) 3.7 (1.7) 6.8 (5.6) 5.5 (3.78) 7.7 (5.7) 4.8 (3.3) 4.3 (2.5) 7.7 (5.7) ** 4.8 (3.2) 7 .1 (5.1) 4.8 (0.9) SCID Depression Dx No Depression 10 (32.3%) 6 (30.0%) 3 (60.0%) 10 (32.3%) 9 (36.0%) 6 (19.4%) 13 (52.0%) 0 (0.0%) 6 (19.4%) 13 (44.8%) 10 (27.0%) 9 (39.1%) Full Remission 14 (45.2%) 12 (60.0%) 2 (40.0%) 14 (45.2%) 14 (56. 0%) 15 (48.4%) 9 (36.0%) 3 (75.0%) 15 (48.4%) 12 (41.4%) 15 (40.4%) 12 (52.2%) Partial Remission 3 (9.7%) 0 (0.0%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 0 (0.0%) 3 (9.7%) 0 (0.0%) 3 (8.1%) 0 (0.0%) Current Depression 4 (12.9%) 2 (10.0%) 0 (0.0%) 4 (12.9%) 2 (8.0%) 5 (16.1%) 1 (4.0%) 0 (0.0%) 5 (16.1%) 1 (3.4%) 5 (13.5%) 1 (4.3%) GAF rating 74.2 (11.4) 76.2 (11.0) 78.8 (11.9) 74.2 (11.4) 76.8 (11.1) 71.9 (11.1) 80.5 (10.4) ** 73.3 (7.9) 71.9 (11.1) 79.4 (10.3) ** 74.2 (11.3) 77.3 (11.3) Note: Column s are defined based on g enotype. Last two columns of maternal and infant groups compare homozygous G carriers with carriers of a minor allele. Last two columns of table compare dyads with concordant genotypes with those dyads that are not concordant. Numbe rs in parentheses are standard deviations, unless denoted as a percentage of the sample. denotes p=. 01 and ** denotes p<.05. Abbreviations: Beck Depression Inventory, 2 nd Edition (BDI II), Postpartum Depression Screening Scale (PDSS), Edinburgh Postnatal Depression Scale (EPDS), Structured Clinical Interview for DSM IV TR (SCID), Diagnosis (Dx), GAF = G lobal A ssessment of F unctioning (GAF) as estimated from SCID interviews.

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21 Procedure Participants enrolled in COMIRB protocol #11 1641 were invited to parti cipate in the current study. If participants chose to take part in this study (COMIRB protocol #14 0851) both mother and child were consented to the current project, allowed a chance to ask any questions they may have about participating, and then asked t o provide a salivary sample. The salivary sample was necessary to determine an individuals' OXTR genotype OXTR genetic information was then analyzed in relation to measures completed while participating in COMIRB protocol #11 1641 The measures recorded i n COMIRB protocol #11 1641 that are relevant to the aims of th is study include d demographic questionnaires, self report measures of depression (i.e. Beck Depression Inventory, 2 nd Edition (BDI II), Postpartum Depression Screening Scale (PDSS) Edinburgh Po stpartum Depression Scale (EPDS), and a Structured Clinical Interview for Diagnosis (SCID IV) infant development as measured by the Bayley Scales of Infant Development, 3rd Edition (BSID III), and dyadic emotional availability as measured by observational coding of an unstructured play session (Emotional Availability Scales ; EAS ). Participants were thanked for participating in this study, but they were not offered financial compensation above and beyond the payment they receiv ed for participating in COMIRB protocol #11 1641 (i.e., $50.00). No additional payment was offered for participating in this study in an effort to ensure that subjects did not feel coerced to provide their genetic information. Saliva/Buccal Cell Collection : A cheek swab/saliva sample was taken from the participant (i.e. mother and child) The sample was collected after the subjects had abstained from eating or drinking for 45 60 minutes to ensure a clean sample was obtained based on the

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22 recommendation of the manufacturer of the buccal cell collection kits, Isohelix. The collection kit (SK 1 Isohelix swab) used wa s essentially an oversized cotton swab (see Appendix B ). After the sample was collected (approximately 60 seconds of swabbing per individual ) the unlinked, numerical value give n to subjects for participating in COMIRB protocol #11 1641 was written on the tube containing the saliva sample T he identity of the participant was protected since there was no link between the numerical value and the identity of the participant. DNA I solation : In order to genotype the participants for the OXTR gene (oxytocin receptor gene) rs53576, it was imperative to first isolate the DNA in the saliva sample (see Appendix C ) Since the DNA is located inside the cells themselves, DNA was extracted ou t of the cells by adding a lysis buffer. A lysis (LS) buffer creates an environment in which the amount of water outside the cell is greater than the amount inside the cell which causes water to move into the cells through a process called osmosis. Throug h this process, the cell eventually bursts open exposing DNA. Five hundred microliters of the LS buffer was added to the tube containing the buccal swab (saliva sample) in order for the osmosis process to occur Although the DNA was exposed at this point, there was still a lot of other biological material floating around including proteins and nucleases (nucleases cleave the bonds holding DNA together, which are unwanted for continued DNA isolation ). In order to remove this material, a solution called prot einase K, or PK solution for short, was added to the tube containing the buccal swab. Th e PK solution "digests" proteins and nucleases while leaving DNA intact by exploiting a fundamental difference between proteins and nucleases and DNA. Proteins and nucl eases have amines, which contain nitrogen, on their molecular structure while DNA has

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23 phosphates (called phosphodiester bonds). The PK solution is attracted to the nitrogen on the proteins and nucleases and dissolves them upon bonding. Twenty microliters o f th e PK solution were added and the tube was vortexed briefly in order to accomplish this separation DNA isolation was continued, either on the same day or after storage (the buccal swab sample and collection tube, with the added LS buffer and PK solutio n, can be stored at room temperature for up to 3 years before continuing with DNA isolation and genotyping procedures) by placing the tube in a 60 ¡ C water bath for one hour ( this step ensures the PK solution has had time to digest the unnecessary protein s and nucleases ) T he resultant solution is removed from the original tube and placed in a 1.5 milliliter centrifuge tube. To this solution a capture buffer, or CT solution, was added to the centrifuge tube containing the sample. Th e capture buffer control s the pH of the solution and enables the DNA to be separated from the rest of the material. After the 400 microliters of CT solution was added, the centrifuge tube was vortexed briefly and then placed in a microcentrifuge to spin at 13,000 rpm for 7 minute s. This process cause s the heavier DNA molecules to go to the bottom of the centrifuge tube. The DNA that ends up on the bottom is called the pellet. The rest of the material /solution wa s removed by pipett e leaving the isolated DNA pellet intact Finally, a re hydration buffer, or TE solution, was added to the isolated DNA. Th e TE solution helps stabilize the DNA and prevent degradation After 150 microliters of this solution was added, the sample was ready for genotyping. OXTR genotyping : Once the DNA w as ready for genotyping, 2 microliters containing 10 nanograms of DNA was removed from the isolated sample and placed in a separate polymerase chain reaction, or PCR, tube. PCR is a biochemical technology in molecular

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24 biology that is used to amplify a sing le or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In this case, the OXTR gene is sequenced through the use of a STEPONE qPCR machine. In order to prepare the DN A sample for qPCR, a master mix containing DNA primers, TAQ polymerase, probes and water was added to the PCR tube containing the DNA sample. The tube was placed into the machine where it initially heat ed up the DNA. Th e heating process "unzips" the doubl e helix DNA strand leaving single DNA strands in the solution through a process called denaturation. The primers in the master mix isolate the portion of DNA containing the OXTR gene. In the case of the OXTR gene it is 348 of the 3 billion base pairs that make up DNA that will be isolated by the primers. TAQ polymerase then replicates these isolated portions of DNA amplifying the OXTR SNP rs53576 The binding of the probes in the master mix indicates which alleles are present in each sample. The alleles pr esent determine the OXTR genotype for SNP rs53576 ( i.e. G/G, G/A, or A/A) for each participant. With 2 probes present o ne only bind s to the G allele and the other only binds to the A allele. Located on the probes are molecules that fluoresce, or simply li ght up, when they bind to specific parts of DNA. In their unbounded state they do not fluoresce as if there is a lens cover over a flashlight. Once they bind to the specified allele ( i.e. G or A) however, they each emit a unique wavelength of light that is measured by the STEPONE qPCR machine. This is done to determine the genotypes of the participants for the OXTR gene SNP rs53576 Measures Self report and structured clinical measure s of depression an assessment of dyadic emotional availability, and an assessment of infant development were collected, in addition

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25 to DNA collection and subsequent OXTR genotyping, as described in the procedures section above. Details of each additional measure that was included in this study are provided below. Assessment of Depression : Self reported symptoms of depression was obtained using the Beck Depression Inventory, 2 nd Edition ( BDI II; A. T. Beck, Steer, & Brown, 1996) Edinburgh Postnatal Depression Scale ( EPDS; Cox, Holden, & Sagovsky, 1987) and the Postpartum Depression Screening Scale ( PDSS; Beck & Gable, 2000) Additionally, a Structured Clinical Interview for D SM IV TR ( SCID ; First, Spitzer, Gibbon, & Williams, 2002) was used to assess current and past psychopathology. BDI II. The Beck Depressio n Inventory, 2 nd Edition (BDI II) is a proprietary self report assessment developed as a revision to the original Beck Depression Inventory (BDI) published by Psychological Corporation (A. T. Beck et al., 1996) The BDI II, like its predecessor the BDI, is a twenty one item self report measure developed to detect symptoms of depression. The BDI II revision made changes reflecting revised diagnostic cr iteria included in the DSM IV for major depressive disorder, i.e. changes made to item location, wording, and time frame (Carvalho Bos et al., 2009; Dozois, Dobson, & Ahnberg, 1998; Ward, 2006) Empirical literature consistently demonstrates that the BD I II is an accurate measure of depression, occurring in the postpartum period or otherwise (Boyd, Le, & Somberg, 2005; Chaudron et al., 2010; Dozois et al., 1998; Smarr & Keefer, 201 1) A review of self report instruments used for PPD found the BDI II to have excellent internal consistency, excellent specificity, acceptable sensitivity, excellent positive predictive value, and good concurrent validity (Boyd et al., 2005)

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26 EPDS. The Edinburgh Postnatal Depression Scale ( EPDS; Cox, Holden, & Sagovsky, 1987) is reportedly the most widely used screening questionnaire for postpartum depression, as it has been translated and used in diverse languages, cultures, and settings. The EPDS is a 10 item Likert type self report measure that purposefully assesses emotional and cognitive symptoms of depression, while excluding somatic symptoms, except for one item related to sleep difficulties, due to the normality of experiencing these symptoms shortly after giving birth. S ystematic reviews of the EPDS have shown that the measure has moderate to good reliability, with a positive predictive value that is estimated to be ~50% and a wide range of validity, sensitivity, and specificity values reported since the scales initial pu blication (Boyd et al., 2005) When using a clinical cutoff score > 12, the authors of the EPDS reported the scale's sensitivity at 86% and the specificity at 78% (Cox et al., 1987) However, despite its widespread use as a PPD screening questionnaire, the EPDS includes symptoms of anxiety (Brouwers, van Baar, & Pop, 2001) and therefore, interpreting results as a pure measure of PPD has been cautioned (Boyd et al., 2005) PDSS The Postpartum Depression Screening Scale ( PDSS; Beck & Gable, 2000) is a 35 item Likert type self report instrument assessing symptomatology within seven distinct dimensions (i.e. sleep and appetite disturbance, anxiety/insecurity, emotional lability, cognitive impairment, loss of self esteem, guilt/shame, and suicidal though ts), that has been validated for detecting the presence of depression in the postpartum period. The PDSS boasts 94% sensitivity and 98% specificity at detecting major depression in the postpartum period when employing a cut off score of 80, and 91% sensiti vity and 72% specificity at detecting major or minor depression in the postpartum period when employing a cut off score of 60 (C. T. Beck & Gable, 2001a, 2001b) Additionally, the PDSS has been found to be valid for use

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27 with individuals of varied cultural and ethnic background s (Zubaran, Schumacher, Roxo, & Foresti, 2010) and the measure s 7 th grade reading level may increase the likelihood that women of limited education are able to accurately complete the questionnaire (Boyd et al., 2005) SCID. The Structured Clinical Interview for DSM IV TR ( SCID; First, Spitzer, Gibbon, & Williams, 2002) is a diagnostic interview intended to specify patients that meet criteri a for major mental disorders (i.e. Axis I disorders) according to the DSM IV TR (American Psychiatric Association, 2000) Several studies have demonstrated that the SCID has good reliability with Kappa values ranging from 0.66 0.93 for diagnosis of major depressive disorder (Lobbestael, Leurgans, & Arntz, 2010; Skre, Onstad, Torgersen, & Kringlen, 1991; Williams et al., 1992; Zanarini et al., 2000; Zanarini & Frankenburg, 2001) The true validity of the SCID is di fficult to ascertain, as the diagnosis of mental illness is largely subjective in nature, depending on the symptoms endorsed by the patient and potentially confirmatory observations made by the clinician or witness. However the SCID was developed in an at tempt to standardize the process of clinical diagnosis made ideographically by trained professionals, and, thus, the SCID is often referenced as the "gold standard" as to which diagnostic procedures are compared (Shear et al., 2000; Steiner, Tebes, Sledge, & Walker, 1995) Assessment of Emotional Availability : The Emotional Availability Scales 4 th Edition (EAS; Biringen, Robinson, & Emde, 199 8; Biringen, 2008) describe and assess six dimensions of dyadic interaction, with four on the adult side S ensitivity, S tructuring, N on I ntrusiveness, and N on H ostility, and two on the child side R esponsiveness to adult and I nvolvement of

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28 adult. An asse t of the EAS is that two dimensions' measure child qualities child responsiveness to the caregiver and the child's involvement with the caregiver, thus capturing not only the adult's side of the relationship, but also the child's side of the relationship. Table 2 briefly describes each domain, with corresponding subscales, included within the EAS and Appendix D provides a more detailed review of the EAS scales and subscales. Although culture affects how parents and children interact with one another, the e mphasis on the global presence of an emotional dyadic connection makes this tool an important one, even in varying cultural contexts. Co investigators who received training and certification by the developers of the EAS rated previously recorded free play sessions that lasted 10 minutes Co investigators completing the EAS coding were blind with respect to maternal self reports of symptoms of depression, clinical diagnosis, and OXTR genotype at the time of assessment and scoring.

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29 Table 2 Emo tional Availability Scales and s ubscale d escriptions ADULT S ensitivity accurate reading of cues and expressing a range of emotions to establish a healthy connection Affect looks at range from balanced and genuine to bl and or depressed/withdrawn (1 7) Clarity of Perceptions awareness of signals and responds appropriately (1 7) Timing Aware of importance of timing (1 3) Flexibility Play is fun and adult is flexible (1 3) Acceptance speaks/acts in respectful ways ( 1 3) Amount of interaction right amount or too little (1 3) Conflict moves conflicts to resolution (1 3) Structuring Positive suggestions and guidance of child Guidance proactive and appropriate variety of suggestions (1 7) Success successful attempts of moving child to higher level (1 7) Amoun t Right amount of structure or too little/none (1 3) Limit Setting appropriate boundaries are set (1 3) Firm in Pressure can remain firm but connected in interaction does not cave easily (1 3) (Non)Verbal Structuring both, one, or neither channel used (1 3) Peer vs. Adult Adult is clearly "older and wiser" (1 3) Non Intrusiveness Being available without being intrusive Following Child Lead has an appropriate spacious quality (1 7) Po rts of Entry waits for optimal breaks to enter interaction (1 7) Command s "Do's" used sparingly (1 3) Adult Talking talking used as dialogue and not overpowering (1 3) Didactic Teaching teaches without ignoring relating (1 3) Interferences Only v erbal and only when necessary (1 3) Feel Intrusive child does or does not indicate the adult is intrusive (1 3) Non Hostility Displays of overt and/or covert hostility Lacks Negativity in words or tone of voice (1 7) Lacks Ridiculing mocking/ri diculing statements or behaviors (1 7) Lacks Threat of Separation use of separation as a threat (1 3) Lose Cool ability to maintain composure during stressful time (1 3) Frightening evidence of physical or verbal assaultive behavior (1 3) Silent am ount of silence in interaction (1 3) Themes play is appropriate but not malevolent (1 3) CHILD Child Responsiveness whether child responds to adult in positive way Affect ranging from upbeat to inappropriate to sad/irritable (1 7) Responsiveness when adult initiates, child responds (1 7) Autonomy right amount of age appropriate seeking (1 3) Physical Positioning seeks or avoids physical contact (1 3) Role Reversal parent like behaviors by child (1 3) Lack of Avoidance attentive to play a nd does not exclude (1 3) Task Oriented appropriate level of focus on object play (1 3) Child Involvement degree to which child involves the adult Elaborative Initiative engages in elaborate way (1 7) Simple Initiative child initiates the engag ement (1 7) Use of Adult Uses adult for emotional exchange and not as a tool (1 3) Lack of Over Involvement negative or over involving behaviors (1 3) Eye Contact appropriate amount to none (1 3) Body Positioning child positions body toward adult ( 1 3) Verbal Involvement involves through talking/babbling (1 3) Note: EAS domains listed in the shaded row, with domain specific subscales immediately following. D irect scores per domain consist of overall impression of subscales with scores rang ing fro m 1 7. Eligible individual subscale scores included in parentheses.

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30 Assessment of Infant Development : The Bayley Scales of Infant and Toddler Development III (BSID III; Bayley, 2005; 2006) was used to assess infant cognitive, language, and social emotio nal development (the motor and adaptive behavior scales were not administered). The items on the Cognitive Scale ( Cog ) of the BSID III assess sensorimotor development, exploration and manipulation, object relatedness, concept formation, memory and other co gnitive abilities. The Language Scale evaluates Receptive Communication ( RC ) and Expressive Communication ( EC ). The RC scale assesses auditory acuity, as well as preverbal behaviors related to vocabulary development, including being able to identify object s and pictures, and otherwise respond to words (Bayley, 2006). The EC scale assesses the child's ability to vocalize, and includes items on babbling, gesturing, joint referencing, turn taking, first words, and object/picture naming. In addition, mothers we re asked to fill out the Bayley Social Emotional Scale ( SE ), which for the study's age range has 17 items designed to assess how well the child has attained social emotional milestones, including self regulation, positive and negative affect, activity leve l, adaptability to change, distractibility, and fear/anxiety (Bayley, 2006). The BSID III exhibits good reliability, with reliability coefficients ranging from .74 to .92 for the Cog RC and EC scales in this study's targeted age group Scale convergence and discriminant valid ity are also reported to be high (Bayley, 2006 ). Clinical psychology doctoral students who received training and supervision by an experienced, licensed clinical child psychologist administer ed the BSID III scales BSID III administra tion was recorded, and administration of the Cog RC EC and SE scales generally took between 30 and 60 minutes to complete BSID III administrators were also blind with

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31 respect to clinical diagnoses and maternal self reports of symptoms of depression at the time of assessment and scoring Data Analysis Plan & Power Data was analyzed using IBM SPSS Statistics version 24 (IBM Corp., 2016) Descriptive statistics (e.g. means, standard deviations, frequency distri butions) were calculated to describe to the sample. Differences in demographic, genotypic, and clinical characteristics were assessed using an independent samples T test as reported in Table 1. The initial hypothese s, i.e. that there is a main effect of O XT R genotypes on maternal depression categorization, observed dyadic emotional availability and infant cognitive development score, were tested using between factors analysis of variance (ANOVA). The broadest hypothesis look ed at dyadic emotional availabi lity in the presence of shared OXTR genotype pairings. A recent study found that in a representative sample of the USA population (n=348), 51.5% of the individuals carried a G/G genotype, 41.4% carried the A/G genotype, and 7.1% carried an A/A genotype (Poulin, Holman, & Buffone, 2012) Given the low overall presenc e of homozygous A OXTR genotypes in the general population and the overall positive finding for prosocial characteristics attributed to homozygous G genotypes categorization of OXTR genotypes into two groups (i.e. those that are homozygous G and those car rying an A allele ) were collapsed during posthoc analyses However, initially, ANOVA analyses tested all possible genotypic pairings to determine underlying significance. With the 3x3 ANOVA structure, the study's 61 dyads obtained a power .78 with an effe ct size of .4 (Faul, Erdfelder, Buchner, & Lang, 2009) Data collection for 61 mother infant dyads represents an acceptable sample size for statistical analysi s to detect a large effect.

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32 R emaining hypotheses tested through additional 3x1 ANOVA analyses had a power .78, based on the study's sample size (Faul et al., 2009) Figure 3 Power b ased on s ampl e s ize for a 3x3 ANOVA When an ANOVA indica ted a statistically signific ant result, an analysis of covariance (ANCOVA) or multiple analysis of covariance (MANCOVA) was utilized to determine if the significant result remained after taking into account cova riates ( e.g. minority status, maternal education mother/infant genotype ) or shared variance amongst dependent variables. Ethical Considerations The Colorado Multiple Institutional Review Board (COMIRB) approved the research study protocol, consent forms HIPAA forms, outcome measures, and adve rtisement materials. Appendix E provides the current and past COMIRB approval documentation for this study (COMIRB protocol #14 0851) In addition to protocol approval, COMIRB require d that the principal investigato r, as well as all study related personnel satisfy the basic education requirements for human subject's research. All r esearchers involved in this study completed the CITI Basic course in human subject protections before taking part in any study related ac tivities. 0 .6 0 .65 0 .7 0 .75 0 .8 0 .85 0 .9 0 .95 0 2 0 4 0 6 0 8 0 1 00 T otal sample size = 0.4 E ffect size f F tests ANOVA: Fixed e ects. special. main e ects and interactions Numerator df = 2. Number of groups = 3. err prob = 0.05. E ect size f = 0.4 P ower (1! err prob)

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33 Risks related to this study protocol were minimal given the non invasive procedures utilized for data collection, and relatively large base of literature investigating OXTR genotypes with no evidence found for adverse outcomes DNA was collected by obt aining buccal cells from saliva and all study materials were de identified and given a unique study identification number prior to analysis. Participants chose to take part in the study and were allowed to stop at any time but all participants compl eted the study after being briefed on the study and supplying informed consent All study related materials could be destroyed if requested by the participant at any time during or after their participation in the study but this was never requested by a p articipant All information collected as part of this study took place with the informed consent and cooperation of the participant, and mothers were required to si gn the consent authorization prior to infant saliva collection. No prior medical records or previous protected health information was requested as part of this study. Additionally, all study related materials were de identified and coded with a unique study identifier. De identified data was retained in a password protected spreadsheet (i.e. SPSS file ). A ll subject data collected in paper form was kept in a locked file cabinet in the office of the principal investigator. Videotape recordings of the 10 minute free play session were retained digitally, but deidentified and coded with the participant 's corresponding study identifier. Digital videotape recordings were stored in a password protected folder on a restricted hard drive

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34 CHAPTER III RESULTS Hypothesis 1: Genotype x Depression The presence of depression in the postpartum period will be r elated to the presence of the G/G OXTR allele sequence in the mother Multiple one way ANOVA's were used to test the initial hypothesis that a homozygous G genotype is related to increased rates of depre ssion. As reported in Table 3 n o significan t dif ferences were found between maternal genotype (i.e. GG, GA, or AA) and maternal depression diagnosis as identified by the SCID (n=56; p=.356), global assessment of functioning as reported on the SCID (n=59; p=.598), or depressive screening score on the BDI II (n=59; p=.454), PDSS (n=60; p=.242) and EPDS (n=59; p=.314). When maternal genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozygous G genotype, maternal depression diagnosis was still not s ignificant based on the results of a Pearson Chi Square test (X 2 (3) =2.913, p=.405). However, further posthoc analysis revealed that there was a significant effect on maternal depression by infant genotype (Table 3 ) A Pearson Chi Square test of independe nce was performed to examine the relation between maternal depression screening score and infant genotype. The relation between maternal depression symptoms and infant genotype was significant (X 2 (1) =4.657, p=.031), with mothers of infants with a homozygou s G genotype more likely to have higher depression screening scores according to the EPDS but not for the BDI II or PDSS Further analysis revealed that there was a lso a significant difference between maternal depression diagnosis on the SCID and infant g enotype as determined by a one way ANOVA (F (2,52) = 4.434, p = .017). A Tukey post hoc test revealed that mothers of infants with a homozygous G genotype were significantly more likely to have

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35 a diagnosis of major depressive disorder than mothers of infant s with a heterozygous genotype (p=.012) Maternal depression diagnosis was not significantly different based on the presence of an infant with a homozygous A genotype, though, this could be due to the low number of infants included in the sample with a hom ozygous A genotype (n=4) When infant genotype groups were collapsed to differentiate those that were carriers of an A allele from those that were homozygous G genotype, maternal depression diagnosis was still significantly more likely to be observed base d on the results of Pearson Chi Square test when infant OXTR genotype was GG (X 2 (3) =8.440, p=.038). Not surprisingly, based on the maternal depression results highlighted above current maternal GAF was also found to be related to infant genotype s uch th at mothers of infants with an A OXTR allele tended to have higher interviewer rated GAF scores (r=.335, n=58, p=.010) Table 3 Depression results by mother and infant genotype Mother's Genotype GG GA AA GG GA/AA Depression Screen BDI II score 12.3 ( 9.8 ) 10.2 ( 6.8 ) 8.4 ( 3.8 ) 12.3 ( 9.8 ) 9.7 ( 6.1 ) PDSS score 63.1 ( 21.5 ) 68.3 ( 25.0 ) 51.9 ( 13.3 ) 63.1 ( 21.5 ) 64.0 ( 22.2 ) EPDS score 6.8 ( 5.6 ) 6.1 ( 4.1 ) 3.7 ( 1.7 ) 6.8 ( 5.6 ) 5.5 ( 3.78 ) SCID Depression Dx No Depression 10 (32.3 % ) 6 ( 30.0% ) 3 ( 60.0% ) 10 ( 32.3% ) 9 ( 36.0% ) Full Remission 14 ( 45.2% ) 12 ( 60.0% ) 2 ( 40.0% ) 14 ( 45.2% ) 14 ( 56.0% ) Partial Remission 3 ( 9.7% ) 0 ( 0.0% ) 0 ( 0.0% ) 3 ( 9.7% ) 0 ( 0.0% ) Current Depression 4 ( 12.9% ) 2 ( 10.0% ) 0 ( 0.0% ) 4 ( 12.9% ) 2 ( 8.0% ) GAF rating 74.2 (11.4 ) 76.2 (11.0 ) 78.8 ( 11.9 ) 74.2 (11.4 ) 76.8 ( 11.1 ) Infant's Genotype GG GA AA GG GA/AA Depression Screen BDI II score 13.2 (10.2) 9.2 (5.6) 7.7 (3.1) 13.2 (10.2) 9.0 (5.3) PDSS score 67.3 (26.5) 58.7 (16.6) 65.3 (13.1) 67.3 (26.5) 59.6 (16.1) EPDS score 7.7 (5.7) 4.8 (3.3) 4.3 (2.5) 7.7 (5.7) 4.75 (3.2) SCID Depression Dx No Depression 6 (20.7%) 13 (56.5%) 0 (0.0%) 6 (20.7%) 13 (50.0%) Full Remission 15 (51.7%) 9 (39.1%) 3 (100%) 15 (51.7%) 12 (46.2%) Partial Remission 3 (10.3%) 0 (0.0%) 0 (0.0%) 3 (10.3%) 0 (0.0%) Current Depression 5 (17.2%) 1 (4.3%) 0 (0.0%) 5 (17.2%) 1 (3.8%) GAF rating 71.9 (11.1) 80.5 (10.4) 73.3 (7.9) 71.9 (11.1) 79.4 (10.3)*

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36 Note: Columns are defined based on Genotype. Last two columns compar e homozygous G carriers with carriers of a minor allele. Numbers in parentheses are standard deviations, unless denoted as a percentage of the sample. denotes p<.05. Abbreviations: Beck Depression Inventory, 2 nd Edition (BDI II), Postpartum Depression Sc reening Scale (PDSS), Edinburgh Postnatal Depression Scale (EPDS), Structured Clinical Interview for DSM IV TR (SCID), Diagnosis (Dx), GAF = G lobal A ssessment of F unctioning (GAF) as estimated from SCID interviews. Hypothesis 2: Genotype x EAS Mothers carrying an A OXTR genotype will demonstrate decreased levels of S ensitivity as measured by the E motional A vailability S cales. A one way ANOVA did not find any significant differences between maternal genotype and direct score of S ensitivity on the dyad ic E motional A vailability S cales (F (2,57) = 1.223, p = .302). Additionally, a separate one way ANOVA did not find any significant differences between infant genotype and direct score of S ensitivity on the EAS (F (2,56) = 1.146, p = .325). However, further r eview of the EAS demonstrated a significant difference between infant genotype and direct score of N on I ntrusiveness (F (2,56) = 4.034, p = .023) s uch that mothers of infants with an A OXTR allele tended to score higher ( i.e., closer to optimal) on the N o n I ntrusiveness scale ( r=.268, n=59, p=.040). Table 4 EAS correlations of direct scores Sensitivity Structuring Non I ntrusiveness Non Hostility Child Responsiveness Child Involvement Sensitivity 1 Structuring .731** 1 Non I ntrusiveness .385** 0.225 1 Non Hostility .503** .581** 0.174 1 Child Responsiveness .375** .427** .316* .286* 1 Child Involvement .578** .530** .387** .410** .637** 1 Note: ** denotes significance at the 0.01 level (2 tailed), and denotes significance at the 0.05 level (2 tailed). However, due to concerns with multicollinearity of the EAS direct scale scores (Table 4 ), a posthoc multivariate analysis of variance (MAN OVA) was performed with each of the

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37 direct scale scores of the EAS ente red as a dependent variable and infant and mother genotype s entered as fixed factor s (i.e. GG vs GA/AA) The results of the MANOVA still demonstrated a significant difference between infant genotype and direct score of N on I ntrusiveness on the EAS (F (1,58 ) = 5.779 p = .020 # 2=.097 ). However as shown in Table 5, when minority status and maternal education were entered into a multivariate analysis of covariance (MANCOVA) infant genotype failed to show significance related to the Non Intrusiveness direct scale score but mat ernal education was significant (F ( 1,59 ) = 14.623 p = 00 0 # 2=.219 ) This suggests that the variance in mean scores of Non Intrusiveness direct scale scores is best accounted for by maternal education, as opposed to infant genotype. Table 5 Non I ntrusiveness direct score MANCOVA result Sum of Squares df Mean Square F Partial Eta Squared Maternal Genotype 3.865 1 3.865 3.712 .0 67 Infant Genotype 3.188 1 3.188 3.062 .056 Minority 1.661 1 1.661 1.595 .030 Maternal Education 15.2 25 1 15.225 14.623* .219 Note: denotes p=.000. Hypothesis 3: Genotype x Infant Performance Infants of depressed mother s who are carriers of an A OXTR allele inherit a protective genetic factor that leads to more optimal performance on the C hild R espon sivene ss direct score on the EAS and cognitive d evelopment score on the BSID III R atings of C hild R esponsiveness on the E motional A vailability S cales A one way ANOVA did not find any significant differences between infant genotype and direct score of Ch ild Responsiveness on the EAS (F (2,56 ) = 1. 359 p = 265 ). Additionally, after collapsing infant genotype groups to differentiate those that were carriers of an A allele from those that were homozygous G genotype, direct scores of C hild R esponsiveness on t he EAS was still not significant based on the results of a Pearson Chi Square test (X 2 (6 ) =7.522, p=.275).

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38 In posthoc analysis, the Child R esponsiveness total, direct, and subscales scores were entered into a MANCOVA that tested associations between mother and infant genotypes and demographic covariates. The total score, direct score, and subscale scores (i.e. Affect R esponsiveness A utonomy, P hysical Positioning Lack of Avoidance and T ask O riented ) were entered as dependent variables and infant / mother g enotypes and concordance were entered as fixed factors while minority status and maternal education were entered as covariates The results of the MAN C OVA demonstrated a significant difference between maternal genotype and Responsiveness subscale score on the EAS (F (1,59 ) = 3.984 p = .0 51 # 2=.071 ) such that mothers that carried an A allele were associated with marginally higher mean (i.e. GA/AA x $ = 5.919; GG x $ = 5.617) scores as observed on the Responsiveness subscale of the Child Responsiveness domain. Additionally, as a covariate highe r maternal education significantly differentiated observed scores on the Child Responsiveness total score (F(1,59 ) = 4.690, p = .035 # 2=.083 ) Autonomy subscale score (F(1,59 ) = 5.058, p = .029 # 2=.089 ), and Physical Positioning subscale score (F(1,59 ) = 4.036, p = .050 # 2=.072 ). C ognitive development, as measured by the Bayley's Scales of Infant Development, 3 rd Edition. A one way ANOVA did not find any significant differences between infant genotype and cognitive development scaled score on the BSID II I (F(2,53 ) = .141, p = .869). Additionally, after collapsing infant genotype groups to differentiate those that were carriers of an A allele from those that were homozygous G genotype, no significant differences were found on the cognitive development scal ed score based on the results of a Pearson Chi Square test (X 2 (9 ) =4.009, p=.911). Figure 4 shows the mean BSID III cognitive development percentile rank by infant genotype.

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39 Figure 4 BSID III percentile rank by genotype Note: BS ID III average percentile ranks shown by genotype for Cognitive, Receptive Communication, and Expressive Communication domains. No significant ass ociations were found between mo ther, infant, or concordant genotypes and BSID III scaled scores. Posthoc ana lysis considered infant and maternal genotype in relation to not only the cognitive development scale of the BSID III, but also the expressive and receptive communication scales. The cognitive, expressive communication, and receptive communication scaled s cores were entered as dependent variables and infant and mother genotypes were entered as fixed factors (i.e. GG vs GA/AA), while minority status and maternal education were entered as covariates. The results of the MANCOVA demonstrated that, in our sample minority status accounted for a significant portion of the variance recorded on the BSID III rec eptive communication scale (F(1,56 ) = 4.522, p = .039), but not for the expressive communication scale (F(1,56 ) = .508, p = .479) or the cognitive development scale (F(1,56 ) = 1.106, p = .298). No significant differences were found in relation to either maternal or infant genotype in regards to the BSID III scaled scores for the cognitive, expressive communication, or receptive communication scales (Figure 4 )

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40 Hypothesis 4: Concordance x EAS N on depressed mother child dyads that are both carriers of the homozygous G genotype will demonstrate significantly higher dyadic emotional availability ratings than dyads that did not meet this criterion Repeated Pearson Chi Square tests showed that there were no significant differences between maternal depression diagnosis and concordant homozygous G genotype with any of the direct scores on the EAS : Sensitivity ( X 2 (5) =3.736, p=.588), Structuring ( X 2 (6) =2.651, p=.851), No n Intrusiveness ( X 2 (6) =.577, p=.997), Non H ostility ( X 2 (4) =.867, p=.929), Child Responsiveness ( X 2 (6) =8.75, p=.188), or Child Involvement ( X 2 (7) =5.792, p=.564). In posthoc analysis, concordance was extrapolated to not only include mother infant dyads th at had a homozygous G genotype but rather any concordant dyadic genotypes (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant) and compared to the results of the EAS A MA NOVA was first conducted to determine if genotype concordance was assoc iated to any of the six direct scores on the EAS Genotype concordance was not statistically associated with the observed variance in me an scores on the Sensitivity Structuring Non Intrusiveness Non H ostility, Child Responsiveness or Child Interaction direct scores included in the EAS. Although the EAS 's Sensitivity direct score seemingly maps onto the earlier reported finding between maternal OXTR and sensitivity reported by Bakermans Kranenburg & Van Ijzendoorn (2008) it should be noted that this prior study's measure of sensitivity was based on observer ratings in three distinct subscales (i.e. supportive presence, intrusiveness, and clarity of instruction) that drew from Ainsworth's conceptualization of social responsiveness (Ainsworth, C., Waters, & Wall, 1978; Egeland, B., Erickson, Clemenhagen Moon, Hiester, & Korfmacher, 1990) Therefore, a separate analysis of the subscales included within the EAS's Sensitivity construct looked at mean differences i n rater scores

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41 based on maternal genotype infant genotype and concordant genotypes No significant differences were found for either maternal genotype or infant genotype on any of the seven distinct subscales of the Sensitivity domain on the EAS. However a between subjects analysis of covariance (ANCOVA) revealed that, for the Affect subscale of the Sensitivity construct, OXTR genotype concordance account ed for a significant proportion of the variance in raters' S ensitivity A ffect score after minority status, maternal education, and mothers' and infants' genotypes had been separately taken into account (F(1,54)=7.167, p=.010, # 2=. 119; Figure 5 ) Additionally, a separate one way ANCOVA demonstrated a statistically significant difference between OXTR genotype concordance on t he Interaction subscale of the Sensitivity construct after controlling for minority status, maternal educa tion, and mothers' and infants' genotypes (F(1,54)=6.193, p=.016, # 2= .105; Figure 5 ) Furthermore, these results held when entering all Sensitivity scale based scores into a MANCOVA to rule out the potential effect of shared variance between scale/subscale scores. For posterity, additional posthoc M ANCOVA's were run for each of the remaining EAS domain's with their respective subscales entered as dependent variables No significant associations were made between OXTR genotype concordance and the subscales of the Structuring Non H ostility, Child Responsiveness or Child Interaction domains of the EAS. However after minority status and maternal education had been separately controlled for, OXTR genotype concordance account ed for a significant proportion of the variance in raters' Non Intrusiveness Following Child Lead s ubscale s core (F(1,54)=4.246, p=.044, # 2=. 072), and in raters' Non Intrusiveness Adult T alking subscale score (F(1,54)=5.767, p=.020, # 2=. 095). In each of the reported findings associating dyadic concordance of genotypes to subscales scores of the Sensitivity and N on I ntrusiveness domains, concordance related to

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42 higher scores on the subscales (Figure 5) Figure 5 Concordance by significant EAS subscale Note: Mean scores shown by genotype concordance for Sensitivity Affect, Sensitivity Interaction Non I ntrusivenes s Adult Talk, and Non I ntrusiveness Follow Child Lead EAS subscales. Sensitivity Affect and Non I ntrusiven e ss Follow Child Lead su bscales scored on a 7 point scale, Sensitivity Interaction and Non I ntrusiveness Adult Talking subscales scored on a 3 point scale. OXTR concordance was associated with increased subscale mean scores for each of the displayed scales (p<.05), after cont rolling for minority status and maternal education.

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43 CHAPTER IV DISCUSSION Oxytocin's purported relationship with depression, prosocial parenting behaviors, and constructs such as empathy, trust, and love make it an important variable to study within the mother infant dyad. This study was designed to determine the OXTR rs53576 genotype for a population of mother child dyads and then analyze the results in relation to measures of maternal depression, infant development, and dyadic emotion al availability. In this way, the study was able to tests some previously reported finding s regarding the effects of maternal genotype, while also going a step further to look at the impact of genotypic concordance within mother infant dyads. The stud y's h ypotheses were formulated based on empirical reports suggesting that the G allele, and in particular homozygous G genotypes, predicts decreased sensitivity to stress, improved social skills, prosocial parenting skills, and increased empathy (Bakermans Kranenburg & Van Ijzendoorn, 2008; Costa et al., 2009; H S Kim et al., 2011; Heejung S Kim et al., 2010; Klah r et al., 2015; Lucht et al., 2009; Riem et al., 2011; Rodrigues et al., 2009; Tost et al., 2010) Provided the above tenants held true for our sample, it was believed that the benefits of prosocial parenting behaviors, decreased stress reactivity, and in creased love, trust, and empathy would provide for a more optimal dyadic emotional availability between mothers and infants. Moreover it was believed that optimal dyadic emotional availability would provide appropriate and timely scaffolding for infant de velopment (Vygotsky, 1962, 1978) However, several reports have also suggested that there may be a relationship between the homozygous G genotype and depression. For instance, t he re is a possib ility that a homozygous G genotype may confer a susceptibility for depression (McQuaid et al., 2013;

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44 R. J. Thompson et al., 2011) and depressive s ymptomatology may suppress the positive effects associated with the homozygous G genotype (Riem et al., 2011) Additionally, the presence of a G allele may increa se the likelihood of offspring developing major depression themselves if raised by a depressed caregiver (R. J. Thompson et al., 2011 ) Given the high prevalence of pregnancy related depression (Gavin & Gaynes, 2005) this study's sample may provide insight into OXTR rs53576's role in maternal depression dyadic emotional availability, and infant development. Null Hypothesis Results T he studies main hypotheses concerning links between OXTR rs 53576 variants and ( 1 ) maternal depression, ( 2 ) maternal dyadic sensitivity ( 3 ) infant performance on the EAS and BSID III, and (4) observed EAS scores in relation to dyadic genotype pairings and maternal depression were not supported by these data Maternal OXTR genotype was not associated with increased rates of depression, as measured by a common P P D screening measure or through a structured cl inical interview. This finding may be an artifact of the particular postpartum study sample or the limited sample size, however, it also casts doubt on to the reliability of a previously reported finding suggesting that the presence of a particular OXTR al lele predicts increased rates of depression (McQuaid et al., 2013; Saphire bernstein et al., 2011) while supporting more recent meta analytic findings raising questions about the association betwee n OXTR rs53576 and psychopathology (Bakermans Kranenburg & van Ijzendoorn, 2014; Li et al., 2015) Within a biopsychosocial context (Engel, 1977) genotypic data only partially accounts for biological factors and do not inherently consider the impact psychological functioning and social environment contribute to the development of psychopathology

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45 The second hypothesis tested whether the predicted negative effects related to the presence of an A OXTR allele were supported with respect to the quality of S ensitivity observed within dyadic i nteractions as measured by the Emotional Availability S cales 4 th Edition (Biringen, 2000) Neither m aternal nor infant genotype alone accounted for a significant difference in total or direct scores of dyadic S ensitivity Due to the observational nature of the EAS and the fact that mother infant interactions took place within a laboratory setting it is possi ble that the assigned scores d id not a ccurately represent the mother infant dyad' s true emotional availability It is also possible that the brief laboratory interactions we used to code emotional availability did not accurately reflect the norm for these dyads Such that the setting and natur e of the study may have inherently pulled for mothers to demonstrate what they believed to be prosocial parenting behaviors and optimal dyadic emotional availability. However, it might again be the case that looking at a single OXTR genotype is too reducti onistic to explain the complex intri cacies enmeshed in dyadic emotional availability between mothers and their infants The third hypothesis attempted to test the idea that infants/children carrying an A allele may inherit a protective factor related to social competence, empathy, and proneness to psychopathology" as hypothesized by Feldman ( 2012c) Within the context of this study, the potential protective factor was operationalized through C hild R esponsiveness as measured by the EAS and infant cognitive development, as measured by the BSID III. Tests revealed no statistical diff erence as a function of infant genotype on either observed C hild R esponsiveness or on the results of the cognitive scale of the BSID III. It may still be true that the A allele "provide[s] a buffer that mitigates some of the effects of maternal depression" (Feldman, 2012b, p. 387) but as operationalized for this study infant genotype

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46 did not predict improved responsiveness or cognitive development in the presence of maternal depression. Moreover, this null finding did not offer support for an early difference in child phenotype based on OXTR genotype, as has been repor ted for adolescents growing up with a depressed mother (S. M. Thompson et al., 2014) The fourth, and final, hypothesis attempted to investigate the role OXTR genotype concordance may have in differentiating observed dyadic emotional availability when mothers are not depressed Give n the purported positive associations with parenting behavior and child social competence, it was believed that mother infant dyads sharing a homozygous G genotype would be observed to provide more optimal interactions. However, in our sample, genotype con cordance did not significantly discriminate between mother infant dyad's observed performance on the emotional availability scales. I t is important to note that only the direct scores for the six emotional availability constructs were included in this hypo thesis' original analysis and as previously discussed the mother infant 's brief 10 minute videotaped interactions were recorded after being provided informed consent while in a laboratory setting. It is possible that dyadic interactions lasting longer in duration or taking place in a more natural environment may provide different results. Post Hoc Analysis Potentially the most noteworthy findings to emerge from this study occurred after rejecting the initial hypotheses, and then further investigating th e role dyadic OXTR genotypes relate to measures of depression and emotional availability. As reported under the results for hypothesis #1 (Genotype x Depression) several unique findings related to infant OXTR genotype were related to maternal depression. For this study's sample, it appears that maternal depression status was related to the infant's genotype using both a standardized

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47 depression screener and a structured cli nical interview. The results demonstrate that mothers, regardless of their own genoty pe are more likely to report symp toms of major depression when their infant had a homozygous G OXTR genotype. Given the increased rates of depression, daily functioning, as measured by GAF scores were understandably also reduced for the mothers of infant s that were homozygous GG carriers. The inverse way to interpret this result would suggest that mothers of infants that carry an A OXTR allele are less likely to report symptoms of depression and demonstrate more optimal daily functioning. Additional ana lyses investigating the role of infant OXTR genotype and dyadic genotype concordance also produced some notable results, as reported under the results for hypothesis #2 (Genotype x EAS) First, mothers were observed to score more favorably on the construct of Non Intrusiveness when their child carried an A OXTR allele. Non I ntrusiveness refers to a mother s ability to be present to their child, both emotionally and physically, without being intrusive (Bi ringen, 2008) Inherent in this conceptualization of nonintrusive parental behavior is a dependency on an infant's reactions to their mother's actions and cues. In this way, the findings demonstrate a potential benefit in dyadic interactions based on infa nt's carrying an A OXTR allele. However, this significant result bec ame nonsignificant when maternal education was entered as a covariate. Still, it is notable that we again see the emergence of a potential benefit for dyads whose infants carry an A OXTR a llele. Another notable finding was discovered while conducting posthoc analysis related to hypothesis #3 (Genotype x Infant Performance) When considering maternal genotype's relation to observed scores on the subscales of the Child Responsiveness domain, mothers that carried an A allele were associated with an infant's higher mean scores as observed on

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48 the Responsiveness subscale of the Child Responsiveness domain. According to the EAS, Child R esponsiveness refers to the infant's reaction to parental bids for engagement, and the expression of pleasure. The focus of this domain is on the emotional and social responsiveness displayed by the infant toward the mother. An optimally responsive child is one who is happy in the presence of the mother and is emotio nally and behaviorally responsive In fact the developer of EAS states that the Child R esponsiveness domain is closest to the attachment theory view of the secure/insecure child (Biringen, 2008) With in this domain, the Responsiveness subscale gauges the child's response to adult imitations In this way, maternal carriers of an A allele in our sample were associated with more optimally responsive infants. T his finding again suggests a potential benefit for the partner in a mother infant dyad when their corresponding partner carries an OXTR A allele. Furthermore, posthoc analysis looked at the relationship genotype concordance within mother infant dyads had on observed EAS scores. The term c oncordance wa s used to refer to the sameness within genotypes collected from mothers and their infants (i.e. GG/GG = concordant, GA/GA = concordant, AA/AA = concordant). E vidence was found while conducting additional analysis on hypothesis # 4 (Concordance x EAS) suppor ting a role dyadic OXTR genotype concordance has on higher observed mean scores on the Affective and Interaction subscale s of the Sensitivity domain and on the Following Child Lead and Adult Talk subscales of the Non Intrusiveness domain on the EAS As con ceptualized by the Emotional Availability Scales, 4 th Edition, the Affect and Interaction subscales are particularly salient to the construct of Sensitivity The EAS manual states "The key characteristic of the sensitivity construct, in our view is affect (Biringen, 2008 p.16 ) because "The child also enjoys interactions with the adult. In other words, the adult cannot

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49 "look" good without the child" (Biringen, 2008 p.17 ) Moreover Non I ntrusiveness refers to the appropriateness of directedness on the part of the mother, providing balanced stimulation when interacting with her child In an optimal non intrusive relationship, there is a lack of over directedness, over stimulation, interference, and over protectiveness. The N on I ntrusiveness scale holds a high respect for the autonomous purs uits of the child. Higher scores on Following the Child Lead and Adult Talk subscales of the Non Intrusiveness domain captures the essence of the EAS's conceptualization of an optimal non intrusive dyadic relationship. Empirical Significance By testing the originally state d hypotheses and performing additional posthoc analysis, this study was able to provide additional evidence for OXTR rs53576 purported role in maternal depression, prosocial parenting or sensitivity, infant development, and dyadic emotional availability. B ased on th is study's significant findings, it is worth considering whether a mother's or infant's genotype or the concordance of dyadic genotypes are partially responsible for driv ing reports of maternal depression or on observed measures of emotional avai lability Klahr et al. (2014, 2015) highlighted the possible importance of evocative gene x environment interactions in interpreting gene associated studies. This study's findings offer tentative support for the idea that OXTR rs53576 genotype analyzed in isolation may not adequately account for outcomes within close social relationships. As highlighted in Feldman's recent review article on The N eurobiology of Human A ttachments (2017 p.87 ) "biobehavioral synchrony, the coordination of biological and behav ioral processes between attachment partners during social contact, is a critical component of human attachments (Feldman, 2012a, 2015, 2016, 2017; Romero Fernandez, Borroto Escuela, Agnati, & Fuxe,

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50 2013) From this p erspective prior reports that investigated the role of maternal OXTR genotype on sensitivity, without consideration for the potential importance of dyadic concordance in their sample's genotype pairings, may be due to a type II error if concordance was no t collected or analyzed. For instance, it could be hypothesized that if the findings of this study regarding the importance of genotypic concordance on measures of Sensitivity on the EAS held true for other samples, then it is possible that Bakermans Krane nburg & van Ijzendoorn's often cited finding linking the maternal GG genotype with increased sensitivity scores (Bakermans Kranenburg & Van Ijzendoo rn, 2008) may vanish. Which, in turn, would inadvertently support Li et al.'s (2015) meta analysis disassociating a link between OXTR rs53576 polymorphism and sociability in the context of close relationships. Alternatively, the results of the two recent OXTR related meta analyses reporting on associations between genotype polymorphism, psychopathology, and sociability may be quite different if concordant genotype analysis was available. Additionally based on the results obtained in this study, it may be increasingly important to consider the infant 's or child 's genotype in the assessment of prosocial parenting, emotional availability, and postpartum depression. In line with the recent conceptualization stressing the coordinated dyadic nature of optimal ox ytocinergic system functioning, the results of this study demonstrate that an offspring s genotype may be related to more or less optimal maternal functioning. Considering the model of biobehavioral synchrony proposed by Feldman (2017) for initiating and m aintaining human attachments, the findings associating child genotype and dyadic concordant genotype pairings with depression status and observed components of emotional availability respectively may

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51 illustrate a small facet of the complexity involved wi thin the biobehavioral underpinnings of the endocrine and neurological systems It also should be noted that within this study, significant findings suggest that the presence of an A allele may impact a partner's depression symptomatology or observed dyad ic emotional availability. In regards to depression status, mothers reported less symptoms and had more optimal daily functioning when their infants carried an A allele. Similarly, mothers were found to display more optimal dyadic emotional availability ra tings on key subscales included within the Sensitivity and Non Intrusiveness domains of the EAS when their infants carried an A OXTR allele Moreover, we found that critical areas of a Child's Responsiveness within the context of dyadic emotional availabil ity was rated more favorably when mother's themselves were carriers of an A allele. An interpretation of this finding may suggest that carriers of an A allele demonstrate increased autonomy which then signals a sense of well being back to their partner fur ther perpetuating more optimal oxytocinergic functioning within a biobehavioral synchrony framework. Additionally, these partner dependent findings may support the notion posited by Feldman (2012a) that the A allele confers a sort of protective factor. Stu dy Limitations Despite the acceptable power of the current study, concern is warranted due to the relatively small sample size and dependence on a large observed effect. There is a chance that results of this study did not adequately describe the main effe cts for the greater population of mother infant dyads in the US, particularly given that ethnic and cultural differences have been noted in the literature as a potential confound for OXTR related studies (Feldman et al., 2016) Additionally, parent characteristics may have influenced observer

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52 report of dyadic emotional availabilit y and behavior (Pauli Pott, Mertesacker, & Beckmann, 2005; Sameroff, Seifer, & Elias, 1982) However, prior studies have demonstrated that mothers tend to provide a valid report of their children (Bates & Bayles, 1984) and a prior study looking at the effects of OXT administration on paternal emotional availability has been published with a slightly smaller sample size (Naber et al., 2010) Also, despite the myriad of studies associating OXTR to human affiliation, prosocial behaviors, and psychopathology, effects are likely due to a complex biopsychosocial proce sses that relies on environmental contingencies and vast biological systems (Benarroch, 2013; Donaldson & Young, 2008; Feldman, 2017) Conclusion Oxytoc in and its single receptor, OXTR, have been well studied since becoming the first human polypeptide hormone sequenced and biochemically synthesized. Oxytocin's role in uterine contractions and mammalian milk production led to findings demonstrating the hormone's role in pair bonding, social af filiation, and attachment. More recently, studies have demonstrated significant relationships between oxytocin levels and OXTR genotypes in relation to prosocial parenting behaviors, measures of empathy, trust, and love, as well as psychopathology. However as we continue to learn more about the complex and multifaceted nature of attachment and social affiliation, researchers have begun to investigate the role biobehavioral synchrony plays. Mother infant dyads are a particularly interesting sample to study these effects, as biopsycho so cial factors directly impact interpersonal relations at a critical point in infant development and dyadic attachment. Results of this study suggest that maternal genotype alone may not be enough to discriminate predictors of co mplex dyadic interactions. Biobehavioral synchrony between both members of the dyad,

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53 and specifically OXTR ge notype concordance, may offer increased insight into the role oxytocin and OXTR play in observable dyadic emotional availability, as well as the gr eater development of pair bonding, attachment, and social affiliation. In the same breath, given the complexity of biopsychosocial factors impacting observable and measureable outcomes of interpersonal interactions, it is important to continue to look at a dditional pathways that may work in parallel to form meaningful interpersonal connections. Moreover, future research should look to replicate previously published findings regarding OXTR rs53576 's purported association with social affiliation, prosocial pa renting, and psychopathology with particular attention on how phenotypes being studied are operationalized, as well as being mindful about how environmental, ecological, and cultural differences can confound study results.

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!)K)*)$I)'=% Bakermans Kranenburg, M. J., & Van Ijzendoorn, M. H. (2008). Oxytocin receptor (OXTR) and serotonin transporter (5 HTT) genes as sociated with observed parenting. Social Cognitive and Affective Neuroscience 3 (2), 128 134. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19015103 Bell, A. F., Carter, C. S., Steer, C. D., Golding, J., Davis, J. M., Steffen, A. D., Connelly, J. J. (2015). Interaction be twe en oxytocin receptor DNA methylation and genotype is associated with risk of postpartum depression in women without depression in pregnancy. Frontiers in Genetics 6 243. doi:10.3389/fgene.2015.00243 Costa, B., Pini, S., Gabelloni, P., Abelli, M., Lari, L., Cardini, A., Martini, C. (2009). Oxytocin receptor polymorphisms and adult attachment style in patients with depression. Psychoneuroendocrinology 34 (10), 1506 1514. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19515497 Kim, H. S., Sherman, D. K., Sasaki, J. Y., Xu, J., Chu, T. Q., Ryu, C., Taylor, S. E. (2010). Culture, distress, and oxytoc in receptor polymorphism (OXTR) interact to influence emotional support seeking. Proceedings of the National Academy of Sciences of the United States of America 107 (36), 15717 15721. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2936623&tool=pmcentrez&rendertype=abstract McQuaid, R. J., McInnis, O. A., Stead, J. D., Matheson, K., & Anisman, H. (2013). A paradoxical association of an oxytocin re cep tor gene polymorphism: early life adversity and vulnerability to depression. Frontiers in Neuroscience 7 128. doi:10.3389/fnins.2013.00128 Roisman, G. I., Booth Laforce, C., Belsky, J., Burt, K. B., & Groh, A. M. (2013). Molecular genetic correlates of infant attachm ent: a cautionary tale. Attachment & Human Development 15 (4), 384 406. doi:10.1080/14616734.2013.768790 L$0*2JAI0+2$= BA)(+7#"*>BD@*#'*/*"%;&+<%<(#0%*')"(8%'#^%0*#"*(8%*8)<+(8/./=;'*(8/(* ,;"7(#+"'*#"*Z/9%&=/"' P _&/"%"N;&:*i*$/"*Qj^%"0++&"1* [\\k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i*$/"*Qj^%"0++&"1*[\\k@L*T+K%$%&1*+(8%&*&%'%/&78*';::%'('*(8/(* #"0#$#0;/.'*K#(8*(8%*RR*:%"+()<%*=/)*N%*=+&%*';'7%<(#N.%*(+*0%<&%''#+"* >-+'(/*%(*/.L1*[\\e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68 APPENDIX B ISOHELIX SWAB BROCHU RE

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71 APPENDIX C INSTRUCTIONS FOR ISO HELIX DNA ISOLATION KITS Isohelix is a division of Cell Projects For swab or DNA isolation queries email: info@isohelix.com www.isohelix.com Molecular Biology Solutions www.cellprojects.com Page 1 of 2 Instructions for Isohelix DNA Isolation kits: DDK 3/DDK 50 Product Details Isohelix Buccal DNA Isolation Kits have been specifically formulated to produce high DNA yield and purity from buccal swabs. The kits hav e been fully optimised at Cell P rojects for use on buccal cell samples and offer reduced handling times, increased DNA yields and many other important technical benefits for their use in manu al, 96 well or other high throughput formats. Key Benefits Kit Contents Isohelix DDK DNA Isolation Kits Catalogue No. DDK 3 DDK 50 Storage temperature Number of preps 3 50 Solution LS (Lysis buffer) 1.5ml 25ml Room temperature Solution PK (Proteinase K) 100 l 1ml 20 0 C Solution CT (Capture buffer) 1.5ml 25ml Room temperature Solution TE (Re hydration buffer) 500 l 1 5 ml Room temperature Storage STORE KITS AT CORRECT TEMPERATURE ON ARRIVAL Isohelix DNA Isolation Kits are shipped at ambient temperature. Please note that on arrival the kit components should be stored according to the table above. The kits are stable up to the expiry date if stored as instructed. See box label for expiry date. Equipment and reagents to be supplied by user Water bath or heating block at 60 0 C Pi pettes with disposable tips Microcentrifuge (with rotor for 2 ml tubes) 1.5ml microcentrifuge tubes Vortexer Before Starting 1. Prepare a waterbath at 60 0 C 2. If a precipitate has forme d in solution LS, warm at 60 0 C for a few minutes 3. Remove the PK solution from the freezer and allow to thaw at room temperature. Note: If a fine white powder precipitate has formed in the PK, vortex the tube immediately prior to pipetting the PK. The precipitate should not block the pipette tip and will dissolve when added to the LS buffer. This precipitate if present has no adverse effect on the activity of the Proteinase K. Technical Assistance If you have any questions regarding the use of this kit or other Isohelix products please contact us by email at info@isohelix .com or for further information visit the website at www.isohelix.com Safety and Use of t he DDK DNA Isolation kits The DDK kits are intended for use by qualified professionals trained in potential laboratory hazards and good laboratory practise. If direct information is not available on any of our compounds this should not be interpreted as an indication of product safety. This kit has been designed solely for research use only Optimised for buccal cells Fast handling times High purity and yield No solvent based chemicals Protocol integrated to swabs Manual or high throughput formats No columns or filtration Less consumables wastage Version 6 Updated November 2011

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72 Isohelix is a division of Cell Projects For swab or DNA isolation queries email: info@isohelix.com www.isohelix.com Molecular Biology Solutions www.cellprojects.com Page 2 of 2 DNA Isolation Protocol Part A DNA Stabilisation 1. Add 500ul LS solution to the tube containing the buccal swab. 2. Add 20 l PK solution to the tube containing the buccal swab and LS solution. Vortex briefly. At this point the DNA is stabilised. You may continue with the DNA isolation or store the stabilised swab in a sealed tube at room temperature for at least 3 years. Part B DNA Isolation 3. Place the tube containing the swab, LS solution and PK solution in a 60 0 C water bath for 1 hour. Vortex briefly. 4. Re set the water bath to 80 0 C if you are using step 1 4 5. Transfer the liquid in the tube (approx. 400 l) into a 1.5ml centrifuge tube using a sterile pipette tip. 6. Optional step to increase yield: If using SK 1 swabs tip the swab head and stick into a sterile 1.5ml centrifuge tube so that the swab head is uppermost. Spin the tube briefly and using a sterile pipette tip add the recovered supernatant to the 400 l collected previously. If using SK 2 swabs spin the SK 2 tube containing the swab head briefly and using a sterile pipette tip add the recovered supernatant to the 400 l collected previously 7. Add 400 l CT solution to the tube (500 l if using the optional step 6 ) Vortex briefly. 8. Place the tube in a microcentrifuge (see tip below ) and spin at approx. 13K rpm for 7 minutes to pellet the DNA. Note the pellet may not be visible Tip: Place the tube with hinge positioned outwards so the liquid can be removed from the opposite side. 9. Remove all the supe rnatant carefully with a pipette tip taking care not to disturb the DNA pellet. 10. Re spin the tube briefly and remove any remaining liquid Note it is important to remove all of the liquid 11. Ad d 150 l TE solution to the tube. This volume may be decreased to as little as 30 l if a higher concentration of DNA is required. 12. Leave for at least 5 minutes at room temperature for the DNA to re hydrate longer if a reduced volume of TE has been used Vor tex briefly. 13. Optional step (May improve lower than expected A260/280 and A260/230 ratios.) Spin tube at 13K rpm for 2 min utes to remove any undissolved particulates and remove supernatant to a clean tube. 14. Incubate the tube at 80 0 C for 5 minutes. Vortex and spin the tube briefly. Note do not use this step if you require double stranded DNA for example restriction digests The DNA sample is now ready for use in downstream applications such as amplification. Store the DNA sample at 4 0 C for short term storage or 20 0 C for long term storage. The expected yield from a buccal swab is 2 to 10 g DNA (10 to 70ng/ l). Our DQC 50 DNA Quality Check Kit is designed specifically to confirm presence of and to test both the quality and quantity of your human DNA by a quick PCR test before you start downstream testing. Other Cell Projects Products Isohelix DNA Buccal Swabs High yields, blood alternative, reproducible, easy to use, different formats for various extraction methodologies. Isohelix DNA Silica Gel Capsules For use with SK 1 swab kits, air dries swab in tube giving extended storage times without loss of stability. Isohelix DNA Isolation and Handling kits DNA stabilising kits for the stable storage of DNA at Room Temperature for long periods : DSK 3/50 DNA quality check by PCR to confirm quality of DNA prior onward experimentation: DQC 50 DNA Release kits quick and easy kit for PCR ready DNA from buccal swabs in under 20 minutes: BEK 50 Isohelix Spin+Collect TM sample recovery devices to increase yields from swabs and other materials: SC/ST 100 PCR Products A range of high quality PCR plastic for 96 well format plates and cap strips Electroporation The HiMaX electroporation cuvettes and buffers maximise molecular electroporation and ele ctrofusion efficiencies for Bacteria, Yeast, Insect, Plant and Mammalian cells.

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73 APPENDIX D EAS DOMAIN AND SUBSC ALE DESCRIPTION Mat ernal Sensitivity refers to a mother's ability to pick up on infant cues, provide warmth and soothe distress, and to be responsive in a variety of situations through quality affective interactions with her child. An optimally sensitive mother creates a gen uine and positive affective environment for the infant, demonstrates a clarity of the infant's needs and expressions, and provides appropriate responsiveness to these needs and emotional expressions. Subscales of the Sensitivity scale include: Affect; Clar ity of perceptions and appropriate adult responsiveness; awareness of timing; flexibility, variety, and creativity; acceptance; amount of interaction; and conflict situations. Structuring involves creating learned opportunities through adequate guidance an d scaffolding. The mother demonstrates successful attempts of following the infant's lead, and sets appropriate limits to discourage maladaptive behaviors. This is done while allowing the child autonomous pursuits and exploration, yet maintaining the "olde r and wiser" framework (Bowlby, 1969) in an optimal parent child relationship. Subscales of the Structuring scale include: provision of guidance; success of attempts; amount of structure; limit setting; remaining fir m in the face of pressure; verbal vs. non verbal structuring; and peer vs. adult role. Non Intrusiveness refers to the appropriateness of directedness on the part of the mother, providing balanced stimulation. In an optimal non intrusive relationship, ther e is a lack of over directedness, over stimulation, interference, and over protectiveness. The non intrusiveness scale also holds a high respect for the autonomous pursuits of the child. Overly involving acts by the mother are alone not be problematic, par ticularly if warranted by the

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74 child's age or developmental abilities, but when they persist in the face of unwelcoming cues by the child, they are considered intrusive. Non intrusiveness differs from structuring in that the latter is focused on guidance an d the former is more about behavioral interference. The subscales of the Non Intrusiveness scale are: Follows child's lead; non interruptive ports of entry; commands, directives; adult talking; didactic teaching; physical vs. verbal interferences; and made to feel intrusive. Non Hostility refers to both overt and covert hostility that can be expressed in language, impatience, or anger. Adult hostility need not solely be directed at the child; rather, any acts of anger, impatience, or boredom in the presenc e of the child is considered hostile in nature. Consistent with the idea of "background anger" and the negative influence this can have on child development (Cummings, 1987) any observable signs of hostility are considered when observing the mother child relationship. The subscal es of the Non Hostility scale include: Adult lacks negativity in face or voice; lack of mocking; lack of threats of separation; does not lose cool; frightening behaviors; silence; and hostile themes of play. Child Responsiveness refers to the infant's reac tion to parental bids for engagement, and the expression of pleasure that accompanies this reaction. The focus is on the emotional and social responsiveness toward the mother. Both affect and responsiveness are considered, and a lack of positive emotional expression or ignoring a mother's invitation to engage is considered non optimal. An optimally responsive child is one who is happy in the presence of the mother and is emotionally and behaviorally responsive to the mother. The Child Responsiveness subscal e is closest to the attachment theory view of the secure/insecure child (Biringen, 2008) The subscales of the Infant/Child Responsiveness scales include: Affect/emotional regulation; responsiveness; a ge appropriate autonomy seeking; positive

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75 physical positioning; lack of avoidance; and task oriented/concentration. Child Involvement refers to extent to which a child seeks to engage the mother in play as well as other means of actively seeking engagement by the mother. In infants, this may be demonstrated through looking and babbling, and in older children this will include both positive verbal and behavioral means to engage the caregiver. Optimal involvement of the parent by the child lacks negative invo lving behaviors, and includes appropriate autonomous exploration, engaging the mother at a development ally appropriate level (Biringen et al., 1998) The subscales of the Child Involvement scale include: Simple initiative; elaborate initiative; use of adult; lack of over involvement; eye contact; body positioning; and verbal involvement.

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76 APPEND IX E COMIRB CERTIFICATE O F APPROVAL Certificate of Approval 29 Sep 2014 Submission ID: APP001 2 Description: Response to Deferral Full Board Initial Application All COMIRB Approved Investigators must comply with the following: For the duration of your protocol, any change in the experimental design/consent and/or assent form must be approved by the COMIRB before implementation of the changes. Use only a copy of the COMIRB approved, stamped Consent and/or Assent Form. The investigator bears the responsibility for obtaining from all subjects "Informed Consent" as approved by the COMIRB. The COMIRB REQUIRES that the subject be given a copy of the consent and/or assent form after it is signed. Consent and/or assent forms must include the name and telephone number of the investigator. Provide non English speaking subjects with a certified translation of the approved Consent and/or Assent Form in the subject's first language. The investigator also bears the responsibility for informing the COMIRB immediately of any Unanticipated Problems that are unexpected and related to the study in accordance with COMIRB Policy and Procedures. Obtain COMIRB approval for all advertisements, questionnaires and surveys before use. Federal regulations require a Continuing Review to renew approval of this project within a 12 month period from the last approval date unless otherwise indicated in the review cycle listed below. If you have a restricted/high risk protocol, specific details will be outlined in this letter. Non compliance with Continuing Review will result in the termination of this study. You will be sent a Continuing Review reminder 75 days prior to the expiration date. Any questions regarding this COMIRB action can be referred to the Coordinator at 303 724 1055 or UCHSC Box F 490. Review Comments: COMIRB noted the Board review and subsequent deferral performed on 30 May 2014. The committee reviewed the PI's written response to deferral and found it to be satisfactory with the exception of the requested itemized changes below. Colorado Multiple Institutional Review Board, CB F490 University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045 303.724.1055 303.724.0990 COMIRB Home Page comirb@ucdenver.edu FWA00005070 [ Phone] [Fax] [Web] [E Mail] [FWA] University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center The Children's Hospital University of Colorado Denver Colorado Prevention Center Investigator: Ryan Asherin Sponsor(s): Subject: COMIRB Protocol 14 0851 Initial Application Effective Date: 19 Sep 2014 Expiration Date: 18 Sep 2015 Expedited Category: 3 Title: Epigenetic effects of OXTR and 5 HTT on maternal depression and child development

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77 Colorado Multiple Institutional Review Board, CB F490 University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045 303.724.1055 303.724.0990 COMIRB Home Page comirb@ucdenver.edu FWA00005070 [ Phone] [Fax] [Web] [E-Mail] [FWA] University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center Children's Hospital Colorado University of Colorado Denver Colorado Prevention Center Certificate of Approval 16-Sep-2015 Investigator: Ryan Asherin Subject: COMIRB Protocol 14-0851 Continuing Review Review Date: 9/15/2015 Effective Date: 15-Sep-2015 Expiration Date: 14-Sep-2016 Sponsor(s): Title: Epigenetic effects of OXTR and 5-HTT on maternal depression and child development Submission ID: CRV001-3 SUBMISSION DESCRIPTION: Response to Minor Modifications Your COMIRB Continuing Review submission CRV001-3 has been APPROVED until the expiration date listed above. The investigator will need to submit this research for Continuing Review at least 45 days prior to the expiration date. Study personnel are approved to conduct the research as described in the documents approved by COMIRB, which are listed below the REVIEW DETAILS section. Please carefully review the REVIEW DETAILS section because COMIRB may have made red-line changes (i.e. revisions) to the submitted documents prior to approving them. The investigator can submit an amendment to revise the documents if the investigator does not agree with the red-line changes. The REVIEW DETAILS section may also include important information from the reviewer(s) and COMIRB staff. COMIRB stamps the approved versions of documents in the top right hand corner. Stamped copies of documents are available for download through COMIRB's electronic submission website, eRA(InfoEd). Click here for instructions on how to retrieve stamped documents. REVIEW DETAILS:

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78 Colorado Multiple Institutional Review Board, CB F490 University of Colorado, Anschutz Medical Campus 13001 E. 17th Place, Building 500, Room N3214 Aurora, Colorado 80045 303.724.1055 303.724.0990 COMIRB Home Page comirb@ucdenver.edu FWA00005070 [ Phone] [Fax] [Web] [E-Mail] [FWA] University of Colorado Hospital Denver Health Medical Center Veteran's Administration Medical Center Children's Hospital Colorado University of Colorado Denver Colorado Prevention Center Certificate of Approval 03-Oct-2016 Investigator: Ryan Asherin Subject: COMIRB Protocol 14-0851 Continuing Review Review Date: 29-Sep-2016 Effective Date: 29-Sep-2016 Expiration Date: 28-Sep-2017 Sponsor(s): None~ Title: Epigenetic effects of OXTR, 5-HTT and irregular cortisol reactivity patterns on maternal depression and child development Expedited Category: 3,7 Submission ID: CRV002-1 SUBMISSION DESCRIPTION: Status: Enrolling Your COMIRB Continuing Review submission CRV002-1 has been APPROVED until the expiration date listed above. The investigator will need to submit this research for Continuing Review at least 45 days prior to the expiration date. Study personnel are approved to conduct the research as described in the documents approved by COMIRB, which are listed below the REVIEW DETAILS section. Please carefully review the REVIEW DETAILS section because COMIRB may have made red-line changes (i.e. revisions) to the submitted documents prior to approving them. The investigator can submit an amendment to revise the documents if the investigator does not agree with the red-line changes. The REVIEW DETAILS section may also include important information from the reviewer(s) and COMIRB staff.