The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

Expert consensus on neurodevelopmental outcomes in pregnancy pharmacovigilance studies.

Background: Exposure in utero to certain medications can disrupt processes of fetal development, including brain development, leading to a continuum of neurodevelopmental difficulties. Recognizing the deficiency of neurodevelopmental investigations within pregnancy pharmacovigilance, an international Neurodevelopmental Expert Working Group was convened to achieve consensus regarding the core neurodevelopmental outcomes, optimization of methodological approaches and barriers to conducting pregnancy pharmacovigilance studies with neurodevelopmental outcomes. Methods: A modified Delphi study was undertaken based on stakeholder and expert input. Stakeholders (patient, pharmaceutical, academic and regulatory) were invited to define topics, pertaining to neurodevelopmental investigations in medication-exposed pregnancies. Experts were identified for their experience regarding neurodevelopmental outcomes following medicinal, substances of misuse or environmental exposures in utero. Two questionnaire rounds and a virtual discussion meeting were used to explore expert opinion on the topics identified by the stakeholders. Results: Twenty-five experts, from 13 countries and professionally diverse backgrounds took part in the development of 11 recommendations. The recommendations focus on the importance of neurodevelopment as a core feature of pregnancy pharmacovigilance, the timing of study initiation and a core set of distinct but interrelated neurodevelopmental skills or diagnoses which require investigation. Studies should start in infancy with an extended period of investigation into adolescence, with more frequent sampling during rapid periods of development. Additionally, recommendations are made regarding optimal approach to neurodevelopmental outcome measurement, comparator groups, exposure factors, a core set of confounding and mediating variables, attrition, reporting of results and the required improvements in funding for potential later emerging effects. Different study designs will be required depending on the specific neurodevelopmental outcome type under investigation and whether the medicine in question is newly approved or already in widespread use. Conclusion: An improved focus on neurodevelopmental outcomes is required within pregnancy pharmacovigilance. These expert recommendations should be met across a complementary set of studies which converge to form a comprehensive set of evidence regarding neurodevelopmental outcomes in pregnancy pharmacovigilance.

Complex chronic conditions among children born to women with schizophrenia.

PURPOSE: Maternal schizophrenia is linked to complications in offspring near the time of birth. Whether there is also a higher future risk of the child having a complex chronic condition (CCC) - a pediatric condition affecting any bodily system expected to last at least 12 months that is severe enough to require specialty care and/or a period of hospitalization - is not known. METHODS: In this population-based health administrative data cohort study (Ontario, Canada, 1995-2018), the risk for CCC was compared in 5066 children of women with schizophrenia (the exposed) vs. 2,939,320 unexposed children. Adjusted hazard ratios (aHR) were generated for occurrence of any CCC, by CCC category, and stratified by child sex, and child prematurity. RESULTS: CCC was more frequent in the exposed (7.7 per 1000 person-years [268 children]) than unexposed (4.2 per 100 person-years [124,452 children]) - an aHR of 1.25 (95% CI 1.10-1.41). aHRs were notably higher in 5 of 9 CCC categories: neuromuscular (1.73, 1.28-2.33), cardiovascular (1.94, 1.64-2.29), respiratory (1.83, 1.32-2.54), hematology/immunodeficiency (2.24, 1.24-4.05) and other congenital or genetic defect (1.59, 1.16-2.17). The aHR for CCC was more pronounced among boys (1.32, 1.13-1.55) than girls (1.16, 0.96-1.40), and of similar magnitude in term (1.22, 1.05-1.42) and preterm infants (1.18, 0.95-1.46). CONCLUSIONS: The risk for a CCC appears to be higher in children born to women with schizophrenia. This finding introduces opportunities for targeted preconception counselling, optimization of maternal risk factors, and intervention to support a vulnerable parent population who will experience unique challenges caring for a child with CCCs.

Prenatal exposure to maternal depression is related to the functional connectivity organization underlying emotion perception in 8-10-month-old infants - Preliminary findings.

Emotion perception is critical for infant's social development. Mother's mood during pregnancy has been associated with infants' emotional developmental risks. Graphtheory analysis was applied on EEG data recorded from 35, 8-to-10-month-old-infants prenatally exposed to high or low depressed symptoms, while viewing happy and sad faces. We found an interaction between group and emotion such that infants exposed to high-depressed-symptoms showed higher modularity - reflecting reduced perceptual-dynamics - for viewing happy emotions compared to sad. The opposite was observed for infants exposed to low-depressive-symptoms. These preliminary findings suggest that prenatal depressive mood may shape early functional organization for viewing emotional faces.

Prevalence and characteristics of adults with fetal alcohol spectrum disorder in corrections: a Canadian case ascertainment study.

BACKGROUND: Individuals with fetal alcohol spectrum disorder (FASD) experience a range of cognitive, affective, and physical deficits following prenatal alcohol exposure. They are thought to be overrepresented in criminal justice settings. However, limited evidence is available to inform prevalence. We sought to estimate the prevalence of FASD in a Northern Canadian correctional population. METHODS: Using an active case ascertainment approach we recruited a representative sample of 80 justice-involved adults (ages 18-40, 85% male) over an 18-month period from 2013 to 2015. Participants completed interdisciplinary clinical assessments comprising medical and psychological evaluations that adhered to the 2005 Canadian FASD Diagnostic Guidelines. RESULTS: We identified a high rate of FASD (17.5, 95% CI [9.2, 25.8%]) in this sample, and this rate could have been as high as 31.2% with confirmation of prenatal alcohol exposure. Most participants in this study presented with significant neurodevelopmental and cognitive deficits in at least two domains of functioning, irrespective of diagnosis, with only five of 80 participants (6.3%) demonstrating no cognitive impairment. CONCLUSIONS: Findings showed disproportionately high estimated FASD prevalence in this representative sample compared to general population estimates in both Canada and the U.S. (2-5%), underscoring the need for improved FASD screening and diagnosis in correctional settings, and education for clinicians working in the justice context. Strengthened health prevention and intervention efforts to support the needs of individuals with FASD outside the criminal justice context are needed.

Infants and adults have similar regional functional brain organization for the perception of emotions.

An infant's ability to perceive emotional facial expressions is critical for developing social skills. Infants are tuned to faces from early in life, however the functional organization of the brain that supports the processing of emotional faces in infants is still not well understood. We recorded electroencephalography (EEG) brain responses in 8-10 month old infants and adults and applied graph theory analysis on the functional connections to compare the network organization at the global and the regional levels underlying the perception of negative and positive dynamic facial expressions (happiness and sadness). We first show that processing of dynamic emotional faces occurs across multiple brain regions in both infants and adults. Across all brain regions, at the global level, network density was higher in the infant group in comparison with adults suggesting that the overall brain organization in relation to emotion perception is still immature in infancy. In contrast, at the regional levels, the functional characteristics of the frontal and parietal nodes were similar between infants and adults, suggesting that functional regional specialization for emotion perception is already established at this age. In addition, in both groups the occipital, parietal and temporal nodes appear to have the strongest influence on information flow within the network. These results suggest that while the global organization for the emotion perception of sad and happy emotions is still under development, the basic functional network organization at the regional level is already in place early in infancy.

Developmental changes in serotonin signaling: Implications for early brain function, behavior and adaptation.

The neurotransmitter serotonin (5-HT) plays a central role in brain development, regulation of mood, stress reactivity and risk of psychiatric disorders, and thus alterations in 5-HT signaling early in life have critical implications for behavior and mental health across the life span. Drawing on preclinical and emerging human evidence this narrative review paper will examine three key aspects when considering the consequences of early life changes in 5-HT: (1) developmental origins of variations of 5-HT signaling; (2) influence of genetic and epigenetic factors; and (3) preclinical and clinical consequences of 5-HT-related changes associated with antidepressant exposure (SSRIs). The developmental consequences of altered prenatal 5-HT signaling varies greatly and outcomes depend on an ongoing interplay between biological (genetic/epigenetic variations) and environmental factors, both pre and postnatally. Emerging evidence suggests that variations in 5-HT signaling may increase sensitivity to risky home environments, but may also amplify a positive response to a nurturing environment. In this sense, factors that change central 5-HT levels may act as 'plasticity' rather than 'risk' factors associated with developmental vulnerability. Understanding the impact of early changes in 5-HT levels offers critical insights that might explain the variations in early typical brain development that underlies behavioral risk.

Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome.

Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2500 fewer steps compared with placebo (Pinteraction=0.04). There were no differences between clonidine and placebo among patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (Pinteraction=0.003) and quality of life (Pinteraction=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions.

Antidepressant use during pregnancy and serotonin transporter genotype (SLC6A4) affect newborn serum reelin levels.

This study was undertaken to determine whether altered early serotonin signaling either via gestational serotonin reuptake inhibitor (SRI) exposure or genetic variations in the serotonin transporter promoter region (SLC6A4) alters levels of reelin, an important glycoprotein in neurodevelopment, in mothers and their neonates. Serum reelin protein expression was quantified by immunoblot from maternal and neonatal blood collected at delivery from women taking either an SRI during gestation or controls. SRI-exposed mothers had higher levels of one reelin fragment, while SRI-exposed neonates had lower total reelin levels, particularly in females and reelin levels differed with SLC6A4 genotype. Lower neonatal reelin levels predicted less time spent sleeping and more irritability during neonatal behavioral assessment on Day 6 of life. Our results suggest that prenatal SRI exposure and the SLC6A4 genotype influences reelin protein expression in both the mother and newborn and that this may be reflected in neonatal behavior.

A register study of the impact of stopping third trimester selective serotonin reuptake inhibitor exposure on neonatal health.

OBJECTIVE: To determine whether risk for adverse neonatal outcomes are reduced by stopping SSRI use before the end of pregnancy. METHOD: Using population health data, maternal health and prenatal SSRI prescriptions were linked to neonatal birth records (N = 119,547) (1998-2001). Neonates SSRI-exposed in the last 14 days (L14) of gestation were compared with infants who had gestational exposure, but not during the last 14 days (NL14). Propensity score matching was used to control for potential confounders (total exposure, maternal health characteristics). RESULTS: Increased risk for neonatal respiratory distress was present where L14 exposure occurred compared with risk where exposure stopped before L14. However, controlling for potential maternal and neonatal confounders, differences disappeared. CONCLUSION: Controlling for maternal illness severity, reducing exposure to SSRI's at the end of pregnancy had no significant clinical effect on improving neonatal health. These findings raise the possibility that some adverse neonatal outcomes may not be an acute pharmacological condition such as toxicity or withdrawal.

Sustained neurobehavioral effects of exposure to SSRI antidepressants during development: molecular to clinical evidence.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are frequently used in the management of antenatal maternal mood disturbances. SSRIs readily cross the placenta and increase central serotonergic tone in the fetus. Given serotonin's key neurodevelopmental role, such prenatal exposure raises concerns about its impact on child development. Preclinical studies report enduring molecular, physiological, and behavioral consequences of developmental SSRI exposure. In humans, sustained developmental outcomes remain largely unstudied, and distinguishing between the effects of prenatal SSRI exposure and the impact of maternal mental illness remains a key challenge.

Depression during pregnancy: the potential impact of increased risk for fetal aneuploidy on maternal mood.

Depression during pregnancy can have serious consequences for families. Indications of fetal aneuploidy can induce maternal stress, a risk factor for depression. Few studies have assessed symptoms of depression in pregnant women soon after they receive results indicating increased risk for fetal aneuploidy. We compared symptoms of depression in women who had increased risks for fetal aneuploidy with two other groups of pregnant women at similar gestational ages: controls, and women taking antidepressant medications (MEDS). Eighty-one women attending the British Columbia (BC) Medical Genetics (MG) Program regarding positive maternal serum screens or ultrasound soft marker findings completed the Edinburgh Postnatal Depression Scale (EPDS). Control (n = 41) and MEDS (n = 41) groups were recruited from the community or the BC Reproductive Mental Health program. A threshold score of 12 on the EPDS was used to calculate percentages of women likely to be depressed. Mean EPDS scores were compared using anova, followed by post-hoc tests. In the control, MG, and MEDS groups, 2.4%, 35%, and 52.4% of women, respectively, scored above 12. Mean EPDS score was significantly higher in the MG group than in the control group (p < 0.0001). These results suggest a place for depression screening in prenatal genetic counseling.

Infant serotonin transporter (SLC6A4) promoter genotype is associated with adverse neonatal outcomes after prenatal exposure to serotonin reuptake inhibitor medications.

Reduced Apgar scores and birth weight, increased risk of respiratory distress, jitteriness and increased tone have been reported in up to 30% of neonates with prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressant medications. In adults, effects of these medications may be related to the genotype for the serotonin transporter (SLC6A4) promoter. In this study we investigated whether SLC6A4 genotype influences the risk for adverse outcomes in neonates with prenatal SRI exposure. Neonatal outcomes including Apgar scores, birth weight, gestational age at birth, symptoms of poor neonatal adaptation and genotype for SLC6A4 were determined in 37 prenatally SRI exposed neonates and compared with 47 non-exposed neonates. Reduced 5 min Apgar scores were observed in exposed neonates and this was moderated by the ss genotype (P<0.001). Birth weight was lower in exposed ls neonates (P=0.008). Risk for respiratory symptoms (respiratory distress and rapid breathing) was higher in exposed neonates with the ll genotype compared to non-exposed neonates (P<0.05) and risk for neuromotor symptoms increased in exposed ss neonates (P<0.026). These relationships remained when controlling for maternal mood during pregnancy, length of gestational medication exposure and gestational age at birth and cesarean section rate. Prenatal SRI exposure was associated with adverse neonatal outcomes and these effects were moderated by infant SLC6A4 genotype. Relationships between polymorphisms and specific outcomes varied during the neonatal period, suggesting that beyond apparent gene-medication interactions, multiple mechanisms contribute to adverse neonatal outcomes following prenatal SRI exposure.

Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events.

Duplications and multiplications of active CYP2D6 genes can cause ultrarapid drug metabolism and lead to therapeutic failure. Multiple functional and non-functional duplication alleles have been further characterized. Duplications were detected by long-range polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and sequence analysis. A PCR fragment encompassing the entire duplicated gene was utilized for detailed characterization. Duplications occurred at 1.3, 5.75, and 2.0% in Caucasian, African American, and racially mixed populations, respectively (n=887 total). Of those 28, 47, and 17% were non-functional CYP2D6*4 x N. Twelve unique duplication alleles were detected: *1 x N, *2 x N, *4 x N, *6 x N, *10 x N, *17 x N, *17 x N[spacer], *29 x N, *35 x N, *43 x N, *45 x N, and a novel non-functional tandem arrangement of a chimeric 2D7/2D6 and *1 gene. All novel duplications except *35 x N were found in African Americans. Accurate identification of gene duplication events is essential to avoid false-positive ultrarapid metabolism assignments and thus, overestimation of predicted activity and increased risk for unwanted adverse events.

Demographic and therapeutic determinants of pain reactivity in very low birth weight neonates at 32 Weeks' postconceptional Age.

BACKGROUND: Management of pain in very low birth weight infants is limited by a lack of empiric knowledge about the multiple determinants of biobehavioral reactivity in infants receiving neonatal intensive care. OBJECTIVE: To examine relationship of early neonatal factors and previous medication exposure to subsequent biobehavioral reactivity to acute pain of blood collection. DESIGN: Prospective cohort study. Methods. One hundred thirty-six very low birth weight (

Relations between behavioral and cardiac autonomic reactivity to acute pain in preterm neonates.

OBJECTIVE: The purpose of this study was to assess relations and concordance between behavioral and physiologic reactivity to pain in preterm neonates at 32 weeks postconceptional age as a function of gestational age at birth. SETTING: Level III neonatal intensive care unit. DESIGN/PATIENTS: The study group comprised 136 preterm neonates (mean [range] birthweight, 1,020 g [445-1,500 g]: gestational age at birth, 28 weeks [23-32 weeks]) separated into three groups according to gestational age at birth as follows: 23 to 26 weeks (n = 48), 27 to 29 weeks (n = 52), and 30 to 32 weeks (n = 36). OUTCOME MEASURES: Reactivity to routine blood collection at 32 weeks postconceptional age was assessed using bedside-recorded behavioral and autonomic measures. Coders who were blinded to the study design scored behavioral responses (facial activity using the Neonatal Facial Coding System, sleep/waking state, and finger splay). Autonomic reactivity was assessed by change in heart rate and spectral analysis of heart rate variability (change in low-frequency and high-frequency power, and the ratio of low-frequency to high-frequency power during blood collection). RESULTS: Facial activity and state correlated moderately with change in heart rate across gestational age groups (r = 0.41-0.62). Facial activity and state did not correlate significantly with change in low-frequency and high-frequency power, or the ratio of low-frequency to high-frequency power (r = 0.00-0.31). Finger splay did not correlate with any autonomic recording (r = 0.03-0.41). Concordance between established biobehavioral measures of pain revealed individual differences. Although some neonates showed high behavioral but low physiologic reactivity, other neonates displayed the opposite reaction; however, the majority displayed concordant reactions. CONCLUSIONS: The study findings confirm the value of measuring domains independently, especially in neonates born at a very young gestational age.

Biobehavioral pain responses in former extremely low birth weight infants at four months' corrected age.

OBJECTIVE: To compare biobehavioral responses to acute pain at 4 months' corrected age between former extremely low birth weight (ELBW) infants and term-born controls. METHODOLOGY: Measures of facial behavioral and cardiac autonomic reactivity in 21 former ELBW infants (mean birth weight = 763 g) were compared with term-born infants (n = 24) during baseline, lance, and recovery periods of a finger-lance blood collection. Further, painful procedures experienced during neonatal care were quantified in both groups. RESULTS: Overall, behavioral and cardiac autonomic responses to the lance were similar between groups. However, the ELBW group seemed to have a less intense parasympathetic withdrawal in the lance period and a more sustained sympathetic response during recovery than the control group. Further, in the recovery period, two behavioral patterns (early recovery and a late recovery) were apparent among the ELBW group. CONCLUSIONS: Biobehavioral pain responses were similar overall between both groups of infants. Subtle differences were observed in cardiac autonomic responses during the lance period and in behavioral recovery among ELBW infants. Whether these findings represent a long-term effect of early pain experience or a developmental lag in pain response remains unclear. The lack of an overall difference runs counter to previously reported findings of reduced behavioral response in former ELBW infants. biobehavioral pain response, premature infants, repetitive pain, heart rate variability.

Prenatal and breast milk morphine exposure following maternal intrathecal morphine treatment.

A case of an infant who had morphine exposure during and following pregnancy while her mother was treated with intrathecal (i.t.) morphine is presented along with a review of the relevant literature. Successful maternal pain management was achieved during pregnancy and while breastfeeding. Minimal maternal serum and breast milk levels in the first 7 postpartum weeks were found. Also, infant sleep and arousal behavior and general development at 2 and 7 months were unremarkable, illustrating the possible safe and efficacious use of i.t. morphine during and following pregnancy. Further work is required to understand opioid pharmacology during breastfeeding, which will be used to develop an empirical approach to breastfeeding and morphine use.

Biobehavioral responses to acute pain in adolescents with a significant neurologic impairment.

OBJECTIVE: To further understand acute pain response in children with a significant neurologic impairment (SNI), we undertook a descriptive hypothesis-generating study of the response to a routine vaccine among adolescents with SNI. DESIGN: Within-subject crossover design. SETTING: Tertiary care facility for children and adolescents with SNI. PATIENTS: Eight adolescents (mean age = 15 years). INTERVENTIONS: Mock and real vaccine injections. OUTCOME MEASURES: Quantitative measures of heart rate, videotaped facial action, Child Facial Coding System (CFCS), and Facial Action Coding System (FACS); observer ratings visual analog scale (VAS) were obtained before, during, and after a mock injection and routine annual influenza vaccine injection presented in a counterbalanced order. RESULTS: VAS scores were significantly higher during the injection phase than during the other time periods; however, there were no significant differences across study time periods when using the other outcome measures. CONCLUSIONS: Although the dampened behavioral and physiologic reactions to an acute noxious stimulus were similar to those of previous work with developmentally delayed children and frail elderly, it remains unclear what underlies the apparent reduced pain response in this setting. These findings have potentially important implications for the daily care of individuals with significant neurologic impairment and illustrate the compelling need tor further study of the unique character of the pain experience in this setting.