humans are social beings who are biologically tuned to form selective and enduring bonds with other individuals . these social bonds provide protection and caregiving and offer comfort during times of distress . a plethora of genetic , epigenetic
, behavioral , neurological , and cognitive processes work in unison to form these meaningful bonds that are essential for human survival .
several studies have examined aspects of the neuroendocrinological basis for social behavior and human bonding .
oxytocin is a neurohormone implicated in the human affiliation system , particularly involving social behaviors , parent - infant bonding [ 3 , 4 ] , and maternal caregiving behavior .
studies in humans [ 68 ] and rodents [ 5 , 9 ] have demonstrated the role of oxytocin in maternal caregiving behavior , with higher levels observed in securely attached mothers after interaction with their infants .
mothers with the highest oxytocin levels during parturition demonstrate more attentive maternal behaviors in relation to the infant , including gaze , vocalization , positive affect , affectionate touch , frequent checking of the baby , and attachment - related thoughts concerning the infant following birth .
also , significant interindividual differences are described in the maternal oxytocin response after interaction with the infant , characteristics which in turn may predict differences in the quality and extent of maternal care [ 11 , 12 ] .
further , mothers with secure attachment styles show an increased oxytocin response following interaction with their infants , exhibiting greater activation of the brain reward regions and the oxytocin - associated hypothalamus / pituitary region .
our group and other researchers have found an association between lower plasma oxytocin levels in the postpartum period and symptoms of adult separation anxiety ( asa ) and depression in mothers ; in addition , these mothers exhibited anxious attachment styles . in the same study ,
asa mediated the association between anxious attachment and depression , while depressed mood mediated the relationship between separation anxiety and low oxytocin levels .
the involvement of oxytocin in regulating the mother 's responsive engagement with her infant has been studied using the still face paradigm ( sfp ) .
sfp is an experimental paradigm initially developed to examine an infant 's reaction to a state of unresponsiveness in the mother , and the paradigm has subsequently been extended to the study of a myriad of related research questions including aspects of mother - infant interaction   and the creation of an emotional challenge for the infant - mother dyad [ 16 , 17 ] . in a study examining change in plasma oxytocin levels following interactions with the infant during the sfp condition
, mothers with a reduced oxytocin response demonstrated a significant decrease in their gaze toward their infants during periods of infant distress , while such changes were not observed in mothers with an increased oxytocin response .
oxytocin , however , may be associated with both positive and adverse outcomes depending on the level of nurturance or adversity in the early environment of an individual .
for instance , recent genetic studies have provided evidence of gene - environment ( g  e ) interactions , such that the snps within the oxytocin receptor gene ( oxtr ) interact with the environment to confer risk for mental disorders and other behavioral traits .
interaction of the oxtr genotypes with positive behaviors such as empathy   and trust   or , conversely , with adverse environments such as exposure to abuse [ 20 , 21 ] shapes either lower or greater risk for depression and anxiety , respectively .
genetic variability within the oxtr gene may modulate risk for psychiatric and behavioral symptoms , and this may be driven by a combination of cumulative genetic effects across the oxtr locus . cumulative genetic risk score across oxtr , cd38 , and avpr1a , previously associated with psychopathology , sociability , and caregiving of the mother ,
has been shown to interact with trauma - exposure in children to predict higher levels of ptsd , anxiety disorders , conduct disorders , and adhd , further supporting the role of oxtr gene - environmental interactions .
the overarching aim of the study was to investigate relations involving blood oxytocin levels , aspects of the oxytocin gene , and maternal caregiving behavior .
specifically , we aimed to evaluate ( a ) the genetic mediators of maternal oxytocin response following a challenging mother - infant interaction task ( i.e. , sfp ) ; test if the same genetic mediators would be associated with ( b ) maternal behavior following a mother - infant separation and interrogate
we hypothesized that mothers with a genetic predisposition to decreased maternal oxytocin response would be more likely to exhibit reduced maternal caregiving behavior , and this relationship would be mediated by separation anxiety .
the study sample comprised 127 participants , selected from a larger pool of pregnant women ( n = 668 ) attending a general hospital antenatal clinic , the details being provided in eapen et al .
briefly , 668 women were screened in pregnancy using the adult separation anxiety questionnaire ( asa-27 ) to assess eligibility for a longitudinal study investigating maternal separation anxiety , oxytocin , and bonding over the transition to parenthood .
the antenatal sample of women was sampled so as to achieve a structured group including a balanced number of participants with and without current separation anxiety symptoms as indicated by the asa-27 .
the study cohort of 127 women had an almost equal number of women with and without asa , with 27 more than the required 100 subjects , to allow for attrition of the sample over time .
from this sample of 127 women , genotypes for oxtr snps were available for a random subset of 96 women that were included in this study .
all analyses described here were performed on this subset of 96 women . at the time of recruitment ,
all women were aged over 18 years , english - speaking , and less than 38 weeks ' gestation with a singleton infant .
women provided consent to participate in a longitudinal study and genetic analysis investigating maternal separation anxiety , maternal bonding , and related experiences .
prenatally ( averaging 30 weeks ' gestation ) , participants completed self - report questionnaires including the adult separation anxiety questionnaire ( asa-27 ) . for the postnatal stage of the study ( averaging 3 months postpartum )
, participants again completed the asa-27 as well as the maternal separation anxiety scale ( msas )   and then took part in the still face paradigm   in addition to providing blood samples for oxytocin measurements and dna for genotyping . 

scores can be summed to yield a total score , with higher scores indicating greater separation anxiety symptom severity .
the measure has sound internal consistency ( cronbach 's alpha of 0.95 ) . to allow comparisons with other studies examining anxiety in pregnancy and the unique contribution of separation anxiety to maternal bonding and related experiences
, participants were allocated to the  separation anxiety group  if they scored over the threshold ( i.e. , total asa-27 score  22 )   for clinically significant symptoms or to the  without separation anxiety group  if they scored below threshold .
the cut - off threshold for determining asa was achieved by comparing the asa-27 with a structured clinical interview for separation anxiety disorder ( adult separation anxiety semistructured interview : asa - si ) that revealed a high level of concordance suggesting that the questionnaire is an adequate alternative measure of asa . 

maternal separation anxiety is characterized by an unpleasant emotional state resulting from a separation experience from her child , manifested by feelings of worry , sadness , or guilt in the mother .
the msas is a 35-item scale composed of 3 subscales assessing a mother 's concerns about separation from her child .
subscale 1 includes items related to feelings of worry , sadness , and guilt surrounding a separation event and attitudes about exclusive maternal care ( maternal care giving behavior as it specifically relates to the infant including separation from the infant ) .
the still face paradigm ( sfp ) [ 7 , 24 ] is a robust and extensively used mother - infant interaction procedure comprising consecutive episodes of  free play  ( 3 minutes ) , maternal  still face  ( 90 seconds ) , and  reunion 
 task for the mother - infant dyad . a trained coder , blind to participants ' questionnaire scores and genetic subtyping , rated maternal behaviors (  maternal sensitivity  ) during the  free play  and  reunion  episodes of the still face paradigm using the global scales for mother - infant interaction .
specifically , maternal sensitivity was coded using the good - poor dimension , rating the extent to which the mother responds to her infant 's cues by appropriately adjusting to the infant 's behavior , responding to infants ' signals , and measuring warmth and acceptance . for the sfp
, ten percent of cases were recoded by a second coder who was also blind to participant scores on all study variables .
the radioimmunoassay ( ria ) method used in this study was developed using independently derived antisera and independently conducted validations and is one of the best available methods for plasma oxytocin estimation .
blood samples were taken before ( t1 ) and after ( t2 ) the still face paradigm , collected into vacutainer tubes , and then centrifuged at 3000  rpm for 10  min .
plasma supernatants were pipetted and stored at 20c until extraction was performed . at the time of extraction , 20  mg heat - activated ( 700  uc ) lichroprep si 60 ( merck ) in 1  ml distilled water was added to each sample , mixed for 30 minutes , and centrifuged .
the pellet was washed with distilled water and 0.01  n acidic acid and then mixed with 60% acetone to elude the neuropeptide , and evaporated extracts were kept at 20c .
the 0.05  ml of assay buffer was added and oxytocin assessed using a highly sensitive and specific radioimmunoassay .
to each aliquot , 0.05  ml antibody and 0.01  ml l - labeled tracer were added and after an incubation period of 3 days unbound radioactivity was precipitated by activated charcoal .
all evaporated plasma extracts to be compared were treated identically to reduce any technical artefacts during the experiment .
the detection limit is in the 0.5  pg / sample range and antiserum cross - reactivity of less than 0.7% .
samples were analyzed at the max plank institute of psychiatry , munich , germany . 

sequencing was performed using miseq reagent kit v2 ( 500 cycles ) 2  250  bp paired end reads .
image analysis was performed in real time by the miseq control software ( mcs ) v2.5.0.5 and real time analysis ( rta ) v1.18.54 , running on the instrument computer .
a total of 12 million reads were generated with > 92% aligning to the human genome .
all data analysis was performed using plink and r. linear regression models in r were fitted for covariates of age and ethnicity .
quality checks removed ambiguous snps , snps with minor allele frequency ( maf ) < 10% , and deviations from hardy weinberg equilibrium ( hwe p value < 0.000001 ) .
this allowed a total of 19 oxtr snps that were used in further analysis . applying a multiple testing correction
the 3 oxtr snps previously reported by feldman et al . , 2014 , were extracted from the imputed filtered dataset . in line with feldman et al .
, a cumulative genetic risk score for oxtr was calculated across rs53576 , rs2254298 , and rs1042778 ( g ) which has been previously associated with parental sensitivity and parent - child reciprocity . for each oxtr snp ,
the genotypes were divided into  high risk  and  low risk  groups as described by feldman et al . , 2014 .
the cumulative genetic risk was computed for each individual by combining the number of high - risk genotypes on each of the snps and summing it across all the oxtr snps .
an extended cumulative genetic risk score across the 3 oxtr snps and rs968389 was calculated using the allele associated with lower oxytocin levels as the risk group .
the clinical cut - off ( 22 ) for the asa-27 ( adult separation anxiety ) was used to group the environmental factor ( e ) adult separation anxiety as binomial . for msas , analysis was performed for each individual subscale as well as for the total msas score .
depending on the variable of interest , linear or logistic regressions were performed for the main and gene - environmental interaction models , after correction for covariates , using standard glm functions in r. coefficients of determination ( adjusted r ) were extracted to indicate the proportion of the variance in the dependent variable explained by the independent variable . to confirm that the snp identified in this study had functional consequences , we queried previous databases and publications for functional evidence .
we used the genotype - tissue expression ( gtex ) project data ( http://www.gtexportal.org/home/ ) and the uk brain expression consortium data ( http://www.braineac.org/ ) to evaluate functional evidence for the snp identified in this study .
these databases allow assessing genetic variation within the genomes by analyzing global rna expression within individual tissues and identifying variations in gene expression that are highly correlated with genetic variation , also known as expression quantitative trait loci , or eqtls .
first , we sought to identify snps within the oxtr gene that significantly regulated maternal oxytocin response ( difference in oxytocin levels before and after the sfp ) .
an association analysis was performed using sequencing data that was available for a total of 19 independent oxtr snps . of the 19
tested snps , one snp within the oxtr , rs968389 , was significantly associated with maternal oxytocin response ( p = 0.0023 , r = 9.1% ) , even after bonferroni correction for multiple testing ( figure 1(a ) ) and after correcting for covariates of age and ethnicity ( p = 0.00259 ) .
given the known association between oxytocin and maternal caregiving behavior , next , we tested the relationship between this snp and maternal behavior specifically related to the care of the infant including separation anxiety as measured by the msas subscales .
the rs968389 was significantly associated with the total msas scores ( p = 0.033 , r = 3.9% ) and specifically with maternal care behavior including when separating from the infant ( p = 0.014 , r = 4.9% ; see figure 1 ) .
no significant association was observed for maternal perception of the child 's response to separation nor maternal feelings about employment - related separation concerns ( p > 0.05 ) . in line with feldman et al . , we calculated the cumulative genetic risk score across the 3 oxtr snps used in the feldman study .
the cumulative genetic risk score of oxtr was not associated with the total msas scores nor with the subscales of maternal separation anxiety from the infant , maternal perception of the child 's response to separation , or maternal feelings about employment - related separation concerns ( p > 0.05 ) .
nevertheless , mothers with a higher oxtr cumulative genetic risk score showed significantly decreased levels of observed maternal sensitivity as measured during the free play and the reunion episodes of the sfp ( p = 0.038 , r = 8.4% ) .
we next questioned whether the association between the genetic risk score and maternal sensitivity was driven by anxiety , using assignment to high or low adult separation anxiety group as measured by the asa-27 .
we tested  evocative  gene - environment interaction   using asa and found a significant g  e association between the oxtr cumulative genetic risk score and adult separation anxiety assignment on observed maternal sensitivity during free play of the sfp ( p = 0.007 , r = 14% , figure 2 ) .
next , we calculated an extended cumulative genetic risk score across the 3 oxtr snps and rs968389 , the snp that we identified to be significantly associated with maternal oxytocin response after the sfp , as shown above .
a significant g  e association was observed between oxtr extended cumulative genetic risk score and adult separation anxiety assignment on the asa-27 on observed maternal sensitivity during free play ( p = 0.035 , r = 5.4% ) of the sfp , similar to that observed above with the cumulative risk score from feldman and colleagues .
in addition , the extended oxtr cumulative genetic risk score was also significantly associated with total msas scores ( p = 0.039 , r = 3.6% ) and the subscale measuring maternal separation anxiety from the infant ( p = 0.022 , r = 3.6% ) ; however , this association was mainly driven by snp rs968389 .
in this study , we interrogated the genetic and environmental modulation of maternal oxytocin response and tested its association with observed maternal behaviors using single and multilocus genetic risk scores within the oxtr gene .
our results suggest that maternal oxytocin response is genetically regulated and we identified a snp within the oxtr gene rs968389 that was significantly associated with changes in oxytocin levels following the sfp .
additionally , this same snp was found to be associated with attitudes about exclusive maternal care and maternal separation anxiety as it relates specifically to the infant , highlighting its role in maternal behavior .
while most mothers are likely to experience some anxiety at separation from their infant , some women experience intense apprehension and concern , resulting in detrimental outcomes for maternal psychological functioning , infant development , and overall family functioning .
as expected , mothers with reduced oxytocin response had increased levels of maternal separation anxiety from the infant . to the best of our knowledge ,
this is the first report indicating this snp in maternal oxytocin response and maternal behavior . using the publicly available gtex database   and the braineac database of the uk brain expression consortium ( http://www.braineac.org/ )
, we found that rs968389 was significantly associated with gene expression of the oxtr gene within the caudate ( basal ganglia ) brain tissue ( p value = 1.7e  8) as well as the frontal cortex ( p value = 8.7e  7 ) among others , providing evidence of functional consequences of the snp ( see supplementary figures 1a and 1b in supplementary material available online at http://dx.doi.org/10.1155/2016/5740365 ) .
moreover , the identified snp rs968389 is also in high ld ( r = 0.9 ) with another snp rs237897 , and this snp was recently reported to significantly influence dna methylation levels for two cpg sites ( p < 0.05 ) within the oxtr gene .
gene regulatory mechanisms are constantly influenced by environmental factors that have the ability to interact with the underlying genotype to dynamically regulate protein activity , thereby influencing oxytocin signaling processes .
we extended the work of feldman and colleagues by using a similar approach and building a cumulative genetic risk score across four snps within oxtr , previously associated with social and maternal behavior .
in addition to classical gene - environment interactions ,  evocative  gene - environment interactions that explain how heritable traits may shape the environment by evoking specific reaction of others and hence the environment provided by others   have been studied in mother - child relationships . for instance , a mother with separation anxiety may be creating a unique environment around her by virtue of her asa and this adverse environment may in turn lead to specific maternal behaviors in response to her infant ; hence we tested evocative gene - environment interactions using asa as the environment .
our results supported a significant g  e association between oxtr cumulative genetic risk score and assignment to high and low adult separation anxiety on observed maternal sensitivity during the free play stage of the sfp , such that mothers with a higher cumulative genetic risk score and with high separation anxiety showed significantly decreased levels of maternal sensitivity compared to mothers with higher cumulative genetic risk score and low separation anxiety .
building upon the published cumulative genetic risk score , we calculated an extended oxtr genetic risk score by adding in the risk information from rs968389 , an independent snp locus which we found to be implicated in maternal oxytocin response and maternal separation anxiety from the infant . using this extended cumulative genetic risk score , we replicated the g  e association between oxtr cumulative genetic risk score and adult separation anxiety ( high versus low ) on observed maternal sensitivity during free play .
furthermore , we identified that the extended oxtr cumulative genetic risk score itself was significantly associated with maternal separation anxiety from the infant ; however this association was driven specifically by rs968389 .
these results suggest a complex mosaic of individual and synergistic genetic effects of oxtr on different aspects of maternal behavior .
while several interesting and novel findings emerge from this study , there are some limitations . the small sample size and
candidate - based approach suggest that it would be important to replicate these findings in larger samples while also performing an unbiased genome - wide scan to identify polygenic predictors of maternal oxytocin response and maternal behavior .
however , the use of a high - risk targeted sample such as the one used for the current study might present an adequate target for highly specialized analysis targeting g  e interactions in well - defined loci . in summary , we identify a novel snp , rs968389 , that is associated with maternal oxytocin response and maternal behavior .
these results contribute to a better understanding of the neurobiological underpinnings of oxytocin and its role in maternal and infant behavior with consequent implications for mother - infant interactions and potential therapeutic interventions that would merit further research .
 we interrogated the genetic modulation of maternal oxytocin response and its association with maternal behavior using genetic risk scores within the oxytocin receptor ( oxtr ) gene . 
 we identified a novel snp , rs968389 , to be significantly associated with maternal oxytocin response after a challenging mother - infant interaction task ( still face paradigm ) and maternal separation anxiety from the infant . performing a multiallelic analysis across oxtr by calculating a cumulative genetic risk score revealed a significant gene - by - environment ( g  e ) interaction , with oxtr genetic risk score interacting with adult separation anxiety to modulate levels of maternal sensitivity . 
 mothers with higher oxtr genetic risk score and adult separation anxiety showed significantly reduced levels of maternal sensitivity during free play with the infant . 
 the same g  e interaction was also observed for the extended oxtr cumulative genetic risk score that included rs968389 . 
 moreover , the extended cumulative oxtr genetic risk score itself was found to be significantly associated with maternal separation anxiety as it specifically relates to the infant . 
 our results suggest a complex montage of individual and synergistic genetic mediators of maternal behavior . 
 these findings add to specific knowledge about genetic regulation of maternal oxytocin response in relation to maternal adjustment and infant bonding through the first few months of life . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion