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Molecular Psychiatry (2008) 13, 65–73 & 2008 Nature Publishing Group All rights reserved 1359-4184/08 $30.00 www.nature.com/mp ORIGINAL ARTICLE Infant serotonin transporter (SLC6A4) promoter genotype is associated with adverse neonatal outcomes after prenatal exposure to serotonin reuptake inhibitor medications TF Oberlander1, RJ Bonaguro2, S Misri3, M Papsdorf1, CJD Ross2 and EM Simpson2,3 1 Early Human Experience Unit, Department of Pediatrics, Centre for Community Child Health Research, University of British Columbia, Vancouver, BC, Canada; 2Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada and 3Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada 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 nonexposed 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. Molecular Psychiatry (2008) 13, 65–73; doi:10.1038/sj.mp.4002007; published online 22 May 2007 Keywords: serotonin transporter (SLC6A4) promoter; neonatal behavior; prenatal exposure to serotonin reuptake inhibitor medications; prenatal maternal depression Introduction Mechanisms that explain why some infants experience poor neonatal outcomes after prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressant medications (selective serotonin reuptake inhibitors and serotonin norepinephrine reuptake inhibitors) remain unclear. Prenatal exposure has been associated with respiratory distress, increased motor tone and feeding/digestive disturbances in approximately 30% of exposed newborns.1 Some studies report that third trimester exposure leads to reduced Apgar scores2 and low birth weight, as well as increased Correspondence: Dr TF Oberlander, Early Human Experience Unit, Centre for Community Child Health Research, Room L408, 4480 Oak St., Vancouver, BC, Canada V6H 3V4. E-mail: [email protected] Received 24 October 2006; revised 7 February 2007; accepted 8 February 2007; published online 22 May 2007 rates of premature birth and increased risk for admission to special care nurseries,3 while other studies have not found similar outcomes.4,5 Concern about these adverse outcomes led the US FDA6 (US Food and Drug Administration) and Health Canada7 to issue warnings in 2004 regarding late pregnancy use of SRIs. Compounding medication-related effects, adverse neonatal behaviors have also been observed following exposure to maternal depression alone8 and therefore identifying SRI-related outcomes has been challenged by the difficulty of distinguishing medication effects from the effects of the maternal disease itself.9 A number of mechanisms have been proposed to explain neonatal adverse outcomes associated with prenatal SRI exposure, including prolonged prenatal central neurotransmitter suppression,10 pharmacological toxicity,11 altered pulmonary vasculature,3 changes in central serotoninergic neurodevelop- Serotonin reuptake inhibitor TF Oberlander et al 66 ment12 and effects of exposure to maternal illness itself.13 Given that SRIs block the reuptake of serotonin (5HT) within the presynaptic cleft via the serotonin transporter (5HTT), this transporter plays a key role as a regulator of serotoninergic neurotransmission and modifier of SRIs effects. Differences in transporter-dependent reuptake efficiency are related to insertion/deletion polymorphisms in the promoter region of the SLC6A4 gene (termed the 5HTT-linked polymorphic region or 5HTTLPR), leading to differential transporter gene expression and clinical differences in SRI efficacy.14,15 SLC6A4 (OMIM16 182138; previous names: 5HTT; serotonin transporter (SERT)) codes for a serotonin reuptake transporter that plays a key role in the regulation of serotoninergic neurotransmission. SLC6A4 transporter 5HT into the presynaptic neuron after serotonin release in brain synapses, which terminates the synaptic actions of serotonin.15,17–19 SRI-mediated blockade of SLC6A4 leads to the accumulation of serotonin in the synaptic cleft and a clinical reduction in depressed mood. A 44 basepair insertion/deletion polymorphism in a region of repetitive sequence in the proximal 50 regulatory region of the SLC6A4 gene has been shown to alter SLC6A4 transcription and activity19–21 leading to differences in SRI efficacy.14,15 The short (s) variant is associated with reduced transcription of SLC6A4 and approximately 50% reduction in serotonin reuptake compared with the long (l) variant.20,22 The ll genotype is associated with significantly better responses to paroxetine and fluoxetine compared with carriers of the ss genotype.14,23–25 Other studies have found the opposite15 or no clinical effect.26 The ss SLC6A4 genotype has also been associated with increased paroxetine-related sideeffects in adults.24,25 Given that SRI clinical effects may vary with SLC6A4 genotype, we sought to examine whether such a relationship exists between this SLC6A4 polymorphism and the risk for adverse neonatal outcomes after gestational SRI exposure. In particular, we aimed to determine whether SLC6A4 genotype moderates the association between SRI exposure and adverse neonatal outcomes. We hypothesized that prenatal SRI exposure would lead to increased risk for adverse outcomes in neonates with two copies of the s allele as a consequence of the reduced SLC6A4 transcription and activity and increased intrasynaptic serotonin levels. Namely, an ss individual would in effect be receiving a higher ‘effective’ dose since there is less serotonin transporter that must be blocked by the SRI leading to an increased incidence of symptoms of poor neonatal adaptation (PNA) and other adverse neonatal outcomes. Methods Subjects With approval from the University of British Columbia Research Ethics Board, Children’s and Women’s Molecular Psychiatry Health Centre of British Columbia Research Review Committee, and informed parent consent, a cohort (n = 98) of mothers was recruited in their early second trimester as part of a study of psychotropic medication use during and following pregnancy. From this cohort neonatal cord and maternal blood samples were obtained for DNA analysis. Of the original 98 mothers approached samples from 14 mothers and infants were not available for analysis because of technical reasons (that is, subject withdrew for personal reasons, inadequate DNA yield, infant blood sample was not obtained at birth) leaving 47 mother– infant samples in the non-medication group and 37 matched samples in the SRI-exposed group. One mother had twins, but only one of the infants participated in this study. None of the mothers took other serotoninergic medications during their pregnancies. Mothers in the SRI treated group, with the exception of two who were started on medication during their second trimester, were already on medication at the time of recruitment and all continued on medication up to the time of delivery (Table 1). Outcomes were compared with healthy mothers not using any medication. Mothers were included if no other psychotropic or antidepressant medications were used during their pregnancy. SLC6A4 genotyping Genomic DNA was extracted from whole-venous blood using the Flexigene DNA Blood Kit (Qiagen, Valencia, CA, USA). The l and s alleles of SLC6A4 were identified as described previously.19 Briefly, PCR was carried out with oligonucleotide primers flanking the polymorphism (corresponding to the nucleotide positions 1416 to 1397 (stpr5, 50 GGCGTTGCCGCTCTGAATGC) and 910 to 888 (stpr3, 50 -GAGGGACTGAGCTGGACAACCAC) of the 50 -flanking regulatory region of SLC6A4 to generate a 484- (s short allele) or 528-bp (l long allele) PCR product. PCR amplification was carried out in a final volume of 30 ml with 50 ng of genomic DNA, 2.5 mM deoxyribonucleotides (dGTP/7-deaza-20 -dGTP = l/l), 0.1 mg of sense and antisense primers, 10 mM Tris– HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 1 U of Taq DNA polymerase. Annealing was carried out at 611C for 30 s, extension at 721C for 1 min and denaturation at 951C for 30 s for 35 cycles. As a quality control 5% of samples were randomly chosen and retested and their genotypes were consistent with previous results. Neonatal health Using previously reported outcomes3,11 neonatal outcomes during two distinct time periods were tabulated. From the immediate newborn period Apgar scores at 1 and 5 min, birth weight, head circumference, length and gestational age at birth were obtained. To distinguish these immediate birth outcomes from those in the later neonatal period, we defined measures of PNA (extending from at least 2 h from birth to time of discharge) as the presence or Serotonin reuptake inhibitor TF Oberlander et al Table 1 67 Maternal and infant demographics characteristics No Exposure (n = 47) mean (s.d.) Maternal demographics characteristics Maternal age at birth (years) Maternal education (years) Delivery (n: vaginal/c section) Maternal mood 2nd trimester HAM-A total score HAM-D total score Edinburgh Postnatal Depression Scale Maternal mood 33 weeks HAM-A total score HAM-D total score Edinburgh Postnatal Depression Scale SRI third trimester dose (n) (median mg (range: min–max)) Paroxetine (18) Fluoxetine (6) Sertraline (5) Venalfaxine (3) Citalopram (5) Maternal SLC6A4 genotype (n) ll ls ss Neonatal characteristics Gestation age at birth (weeks) Birth weight (grams) Small for gestational age (X38 weeks and < 2500 g at birth) (n) Birth length (cm) Head circumference (cm) Sex (M:F) SRI exposure during pregnancy (no. of days) Apgar score at 1 min Apgar score at 5 min Neonatal SLC6A4 genotype (n) ll ls ss SRI Exposure (n = 37) mean (s.d.) 32.70 (4.96) 17.38 (3.07) 35/18 31.84 (4.77) 15.3 (2.45) 26/15 7.30 (7.42) 6.33 (7.34) 5.86 (6.25) 14.16 (7.44)* 12.65 (6.48)* 10.41 (6.16)* 5.79 (4.93) 3.77 (4.44) 4.91 (4.79) 11.43 (7.50)* 9.43 (6.03)* 8.30 (4.53) NA NA NA NA NA 13 26 8 40.14 (1.18) 3605.94 (517) 1 52.09 (2.95) 35.04 (1.30) 24:29 0.00 (0.00) 8.13 (1.36) 9.06 (0.48) 14 22 11 27.5 35 100 75 30 (2.5–45) (10–50) (50–175) (37.5–150) (20–40) 13 14 9 39.34 (1.49)* 3404.95 (506.77) 3 50.85 (2.52) 34.55 (1.34) 19:22 223.95 (74.67) 7.54 (1.46) 8.70 (0.85)* 14 16 7 Abbreviations: HAM-A, Hamilton Rating Scale for Anxiety; HAM-D, Hamilton Rating Scale for Depression; NA, not applicable; s.d., standard deviation. *P < 0.05 for differences between SRI and control groups. absence of bradycardia ( < 100 b.p.m.), tachycardia ( > 160 b.p.m.), increased neuromuscular tone, jitteriness, respiratory distress (nasal flaring, indrawing, grunting), tachypnea ( > 60 min), hypoglycemia ( < 3.3 mmol/l) or hyperglycemia ( > 7.0 mmol/l). While temperature instability has been previously reported, it was not quantified as an outcome in this study because of the lack of clear definitional parameters in the literature.1,3 Occurrences of these outcomes were obtained from a chart review by two research nurses blinded to exposure group status and genotype. Maternal mood During the pregnancy, maternal mood was assessed at the time of study enrollment (approximately 26–28 weeks) and again at 33 weeks gestation using three instruments. The Hamilton Rating Scale for Depression (HAM-D)27 is a 21-item clinician administered scale that measures the severity of depression in adults. Scores on this scale have a possible range of 0–63, with higher scores being associated with higher levels of depression in the patient. Scores ranging from 0 to 7 suggest no or minimal levels of depression, 8–17 indicate mild depression, 18–25 suggest moderate depression, and scores of 26 and above are associated with severe depression. The Hamilton Rating Scale for Anxiety (HAM-A)28 is a 14-item clinician administered scale that measures the severity of anxiety. Total scores on this scale have a possible range of 0–56, with higher scores being associated with higher levels of anxiety in the patient. Scores ranging from 0 to 7 suggest no or minimal levels of anxiety, 8 to 17 indicate mild anxiety, Molecular Psychiatry Serotonin reuptake inhibitor TF Oberlander et al 68 18 to 25 suggest moderate anxiety and scores of 26 and above are associated with severe anxiety. The Edinburgh Postnatal Depression Scale,29 is a 10-item, patient-rated instrument used to assess symptoms of depressed mood in both pre- and postnatal settings. Statistical analysis Factorial analyses of variance were used to examine group (exposed vs non-exposed) and genotype differences in outcomes, as well as the interaction between exposure and genotype to assess the role of SLC6A4 genotype as a possible moderator of exposure. Data are presented as raw data. w2 and hierarchical logistic regression models were used to determine whether adverse neonatal outcomes would be predicted by SLC6A4 genotype. Effect sizes (Z2) were calculated and are presented in the text below. Results Demographic characteristics Maternal and neonatal demographic characteristics and maternal SRI medication use are presented in Table 1. In the SRI treatment group, levels of depression and anxiety symptoms during pregnancy were significantly higher (all P’s < 0.001). SLC6A4 genotype ratios were distributed according to Hardy– Weinberg equilibrium for all 176 subjects (85 infant and 91 maternal) together (allele frequency l = 56% and s = 44%; and genotype frequency: ll = 32%, ls = 47%, and ss = 20%), as well as mothers and infants separately (Tables 1 and 2). As expected, the allele frequency and genotype distribution was not different between the exposed and non-exposed mothers. Mean daily dose or length of prenatal SRI Table 2 exposure did not vary significantly between genotypes (Table 2). Immediate neonatal outcomes SRI exposed infants were born earlier (F = 6.16, P = 0.015, Z2 = 0.07) and had lower Apgar scores at 1 (F = 4.79, P = 0.032, Z2 = 0.06) and at 5 min (F = 11.23, P = 0.001, Z2 = 0.13) compared to nonexposed infants (Table 1). At 1 min, the association between SRI exposure and differences in Apgar scores was not moderated by SLC6A4 genotype (F = 2.25; P = 0.112, Z2 = 0.05), while at 5 min there was a significant interaction between SRI exposure and SLC6A4 genotype (F = 3.28, P = 0.043, Z2 = 0.08; Figure 1). Simple effects tests revealed that neonates with the ss genotype had significantly lower 5 min scores, but only when they had prenatal SRI exposure (F = 12.43, P < 0.001, Z2 = 0.24), controlling for maternal mood during pregnancy, cesarean section rate and gestational age at birth. Interestingly, reduced Apgar scores were primarily due to reduced respiratory effort subscale scores at 1 and 5 min (exposure vs no exposure: 1.48 vs 1.72; P = 0.062 and 1.81 vs 1.99; P = 0.009, at 1 and 5 min, respectively). There was also a significant interaction between SRI exposure and SLC6A4 genotype on birth weight, such that exposed babies had a significantly lower birth weight among neonates with the ls genotype (F = 7.45, P = 0.008, Z2 = 0.19; Table 2). These effects remained significant when controlling for gestational age at birth, maternal pregnancy-related mood and length of prenatal SRI exposure. Interestingly, when controlling for gestational age, birth weight was higher in ss exposed neonates compared with non-exposed neonates (F = 4.50, P = 0.037, Z2 = 0.29). Infant outcomes and SLC6A4 genotype: means (s.d.) SLC6A4 genotype Duration of prenatal SRI exposure (days) Maternal mean daily dose of medication (z scorea) Gestational age at birth (weeks) Birth weight (g) Birth length (cm) Head circumference (cm) Length of newborn stay in hospitals (h) ll ls ss No exposure (n = 14) SRI exposure (n = 14) No exposure (n = 22) SRI exposure (n = 16) No exposure (n = 11) SRI exposure (n = 7) NA 240 (57) NA 206 (92) NA 231 (62) NA 0.181 (0.138) NA 0.299 (0.283) NA 0.153 (0.101) 39.8 3583 52.3 34.8 51.0 (1.45) (594) (3.80) (1.49) (22.2) 39.4 3416 50.1 34.3 61.3 (1.48) (509) (2.27) (1.34) (27.9) 40.3 3691 51.9 35.2 48.6 (1.03) (455) (2.70) (1.28) (25.6) 39.3 3239 50.5 34.5 61.3 (1.61) (549)* (2.42) (1.17) (40.9) 40.3 3465 52.3 35.0 46.9 (1.09) (545) (2.40) (1.14) (27.6) 39.4 3763 53.1 35.2 62.1 (1.44) (367) (2.28) (1.68) (21.4) Abbreviations: NA, not applicable; SRI, serotonin reuptake inhibitor. *P < 0.05 for differences between exposure and non-exposure within genotypes. a A composite drug dosage ‘z score’ was tabulated for drug dosage to account for multiple drugs each with varying drug dose ranges. Molecular Psychiatry Serotonin reuptake inhibitor TF Oberlander et al a b 1 Minute Apgar 9.5 9 Apgar Score 9 Apgar Score 69 5 Minute Apgar 9.5 8.5 ll ls ss 8 7.5 7 8.5 * ll ls ss 8 7.5 7 6.5 6.5 6 6 No exposure No exposure SRI exposure SRI exposure Figure 1 Apgar score, SLC6A4 genotype and SRI exposure. At 1 min Apgar (a) score was not moderated by SLC6A4 (F = 2.25, P = 0.112), while at 5 min, (b) there was a significant interaction between SRI exposure and SLC6A4 genotypes *(F = 3.28, P = 0.043). Proportions of Neonates with PNA symptoms 50 * * Proportions (%) 40 No exposure (n=47) SRI exposure (n=37) 30 20 10 0 Tachycardia (>160 bpm) Bradycardia (<100 bpm) Tachypnia (> 60 min) Respiratory Distress Jitteriness Increased Motor Tone Hypoglycemia Hyperglycemia (< 3.3 mmol/l) (>7.0 mmol/l) Neonatal Outcomes *differences in proportions of SRI exposed symptomatic neonates compared with non exposed neonates, Pearson’s X2 P < 0.05, controlling for 3rd trimester maternal mood Figure 2 Proportions of neonates with symptoms of poor neonatal adaptation (PNA). Gestational age at birth was not moderated by SLC6A4 genotype (P > 0.250). Late neonatal outcomes Beyond the immediate neonatal period (that is, at least greater than 2 h of life), the incidence of PNA were significantly increased in SRI exposed infants as reflected by respiratory distress (indrawing, tugging), jitteriness at rest and increased muscular tone (Figure 2). When maternal mood was included as a covariate in logistic regression models, SRI exposure only significantly predicted respiratory distress and jitteriness (both P’s < 0.010, odds ratios = 9.3 and 10.6, respectively). Moreover, hierarchical w2 tests (see Table 3) revealed that SLC6A4 genotype moderated the effect of prenatal SRI exposure, as exposed infants with an ll genotype were at increased risk for rapid breathing ( > 60 min; w2 = 5.60, P = 0.018), respiratory distress (w2 = 4.09, P = 0.043) and jitteriness (w2 = 6.09, P = 0.014). Likewise, exposed infants with an ss genotype were at increased risk for jitteriness (w2 = 4.92, P = 0.026) and abnormal tone (w2 = 5.66, P = 0.017), while those with an ls genotype were also at increased risk for respiratory distress (w2 = 6.16, P = 0.013) compared with non-exposed neonates with the same genotypes. Cesarean delivery was not a significant covariate. Discussion Prenatal SRI exposure was associated with adverse neonatal outcomes, consistent with numerous previous reports;2,3,10,11 however, these effects were moderated by infant SLC6A4 genotype. Specifically, prenatal SRI exposure was associated with reduced 1 and 5 min APGAR scores, but only the latter was associated with a genotype-SRI exposure interaction. In the immediate newborn period lower 5 min Apgar Molecular Psychiatry Serotonin reuptake inhibitor TF Oberlander et al 70 Table 3 Frequency (%) of PNA symptoms by SLC6A4 genotype and SRI exposure SLC6A4 genotype Tachycardia ( > 160 bpm) Bradycardia HR ( < 100 bpm) Tachypnea breathing ( > 60 min) Respiratory distress Jitteriness Increased motor tone Hypoglycemia ( < 3.3 mmol/l) Hyperglycemia ( > 7.0 mmol/l) ll ls ss No exposure (n = 14) SRI exposure (n = 14) No exposure (n = 22) SRI exposure (n = 16) No exposure (n = 11) SRI exposure (n = 7) 14.3 7.1 14.3 14.3 0.0 0.0 7.1 0.0 57.1 0.0 57.1* 50.0* 35.7* 14.3 21.4 7.1 13.6 4.5 13.6 9.1 9.1 0.0 9.1 0.0 25.0 0.0 25.0 43.8* 25.0 0.0 25.0 0.0 27.3 0.0 27.3 9.1 9.1 0.0 9.1 0.0 14.3 0.0 14.3 42.9 57.1* 42.9* 14.3 14.3 Abbreviations: PNA, poor neonatal adaptation; SRI, serotonin reuptake inhibitor. *Compared with no exposure on Pearson’s w2 (all P < 0.05). scores were observed in exposed neonates with the ss SLC6A4 genotype. Lower birth weight was observed in exposed neonates with the ls genotype, while an increased birth weight was also observed in exposed ss neonates, even when accounting for gestational age at birth. In the later neonatal period, neonates with ll genotype carried an increased risk for respiratory distress and tachypnea, while the ss neonates had an increased risk for jitteriness and increased muscular tone. Neither head circumference nor the incidence of hypo/hyperglycemia or tachycardia was affected by SRI exposure or SLC6A4 genotype. Moreover none these outcomes were related to the effects of exposure to depressed or anxious prenatal maternal mood, having a cesarean delivery, gestational age at birth or length of prenatal medication exposure. The number of infants with a given medication exposure was small and therefore we were not able to determine whether risk for a particular gene exposure interaction was associated with a specific SRI. Further, birth weight was also affected, particularly in ls and ss exposed neonates. While reduced birth weights have been reported following prenatal exposure (consistent with the ls genotype), the increased birth weight in ss neonates is a new finding with no mechanism apparent and will have to be studied further. It remains possible that an increased risk for respiratory distress was also associated with the ss allele, but due to our small sample size of ss neonates we may have lacked the power to demonstrate this relationship. Importantly, the SRI dosage in the treatment group was consistent with typical doses used in clinical practice,30 and was assumed to be adequate given the maternal mood ratings during the second and third trimesters. A number of pharmacological and neurotransmitter related mechanisms have been proposed that may account for poor neonatal outcomes following prenatal SRI exposure. These include pharmacological factors related to varying half lives, pharmacologiMolecular Psychiatry cally active metabolites31 and possible competitive inhibition with other medications.11 Paroxetine’s high potency for inhibition of 5HT reuptake and affinity for muscarinic receptors may lead to adverse neonatal outcomes once medication exposure ceases, suggesting a ‘discontinuity condition’ as proposed by Moses-Kolko.1 Alternatively some of our findings could also represent a condition analogous to the ‘serotonin syndromes’ observed in adults32 associated with increased intrasynaptic serotonin (leading to increased postsynaptic serotonin receptor stimulation), resulting in nausea, diarrhea, agitation and insomnia.32–34 Possible altered regulation of noradrenergic and dopaminergic systems have also been suggested.35 While many of these processes have not been confirmed as a mechanism in prenatally exposed neonates, there are reports of symptoms that might support some of these as possible mechanisms in exposed neonates.3,10,36,37 Our findings appear to illustrate a biphasic pattern of adverse outcomes that may reflect the presence of both a ‘discontinuation’ and a ‘serotonin syndrome’ condition depending on neonate genotype and the time interval from birth during which the infant symptom was observed, as well as the specific symptom itself. In the immediate postnatal period (reflecting prenatal conditions) we observed reduced Apgars at 1 and 5 min (primarily driven by reduced 1 and 5 min respiratory effort scores), particularly in ss neonates. Such neonates would have experienced long-term blockade of 5HT reuptake, leading to chronic high levels of intrasynaptic serotonin (Figure 3a) and coupled with increased postsynaptic receptor sensitivity could lead to a condition analogous to a ‘serotonin syndrome’ condition as observed in adults.32,33 It is conceivable that during this immediate newborn time, the combination of SRIinduced 5HT transporter blockade (leading to reduced neuronal 5HT and increased intrasynaptic 5HT), and a less efficient ss genotype (that is, reduced Serotonin reuptake inhibitor TF Oberlander et al a 71 Possible effects of prolonged prenatal SRI exposure Presynaptic neuron 5HT Transporter (intramembrane) Intrasynaptic 5HT reuptake via transporter SRI exposure: blocks 5HT reuptake 5HT 5HT Transporter blockade leading to chronically high prenatal intrasynaptic 5HT levels 5HT 5HT 5HT 5HT 5HT 5HT 5HT Postsynaptic neuron 5HT postsynaptic receptors b Possible increased postsynaptic 5HT receptor sensitivity following prolonged prenatal SRI exposure Immediate Newborn Period: Continued SRI exposure and SLC6A4 genotype and neonatal behavior ll genotype: (Efficient gene transcription) ss genotype: (Inefficient gene transcription) SRI blocked 5HT reuptake 5HT Transporter (intramembrane) 5HT SRI blocked 5HT reuptake 5HT 5HT 5HT “Typical” Apgar Scores (ll genotype): “relative” abundance of 5HT transporter able to ”compensate” for increased intrasynaptic 5HT secondary to SRI exposure and increased post synaptic 5HT receptor sensitivity c 5HT 5HT Increased 5HT postsynaptic receptor sensitivity Reduced Apgar Score (ss genotype): “reduced” 5HT transporter leads to less 5HT reuptake, an “overabundance” of intrasynaptic 5HT and increased post synaptic 5HT receptor sensitivity Later Newborn Period (> 2 hrs of life): Diminishing SRI exposure, SLC6A4 genotype and neonatal behavior ll genotype: (Efficient gene transcription) ss genotype: (Inefficient gene transcription) Diminishing SRI levels and 5HT reuptake blockade 5HT Transporter (intramembrane) 5HT Diminishing SRI levels and 5HT reuptake blockade 5HT 5HT 5HT Respiratory Distress (ll genotype): “relative” abundance of 5HT transporter transcription leads to relative''deficiency'' of intrasynaptic 5HT, but with continuing increased post synaptic 5HT receptor sensitivity could still lead to respiratory symptoms Figure 3 5HT 5HT 5HT 5HT 5HT 5HT 5HT Increased 5HT postsynaptic receptor sensitivity 5HT 5HT Neuromotor irritability (ss genotype): reduced 5HT transporter transcription leads to less 5HT reuptake and relative “overabundance” of intrasynaptic 5HT, and coupled with continued increased post synaptic 5HT receptor sensitivity, could lead to increased neuromotor symptoms Hypothetical schema for possible interactions between SLC6A4, 5HT, SRI and neonatal outcomes. expression of the transporter gene and less 5HT reuptake), could result in even greater intrasynaptic 5HT levels (Figure 3b). Namely, there would be less ‘target’ serotonin for SRI-mediated blockade, leading to reduced 5HT removal and relatively increased 5HT in the synapse. Alternatively, the increased efficiency Molecular Psychiatry Serotonin reuptake inhibitor TF Oberlander et al 72 associated with the SLC6A4 ll genotype might ‘compensate’ for the effects of increased intrasynaptic 5HT, thereby ‘protecting’ the neonate from the effects of excess intrasynaptic 5HT associated with prenatal exposure. This might be analogous to the improved clinical responsiveness to paroxetine and fluoxetine in adults with the ll genotype25 compared to those with the ss genotype.14,23–25 Other studies have found the opposite15 or no clinical effect at all.26 Moreover, Murphy et al. (2004) reported that adults with the ss genotype experienced significantly more severe adverse events and tolerated lower doses of paroxetine.24,25 In contrast, during the later postnatal period (at least 2 h from birth) we observed a variety of symptoms characterized by respiratory distress and increased neuromotor tone. During this time SRI exposure and suppression of 5HT reuptake presumably diminishes resulting in relative decreased levels of intrasynaptic 5HT. However, such decreased 5HT coupled with continued supersensitive upregulated postsynaptic receptors could lead to a situation reflecting a ‘discontinuation’ condition seen in adults.38 In the exposed ll neonate, where there might be a relative abundance of transporter (that is, efficient SLC6A4 transcription), leading to improved neonatal reuptake and possibly more ‘normalizing’ levels of intrasynaptic 5HT. However in the continued presence of increased postsynaptic 5HT receptor sensitivity this might still lead to increased risk for respiratory distress. In contrast, the neonate with ss genotype and SRI exposure, may be unable to ‘efficiently’ uptake 5HT presynaptically, resulting in a ‘relative excess’ of intrasynaptic 5HT leading to increased risk for jitteriness and muscle tone. However, precise mechanisms that account for each condition remain a matter for future studies. Reduction of Apgar scores, birth weight and an increased risk for symptoms of PNA have been observed in approximately 30% of neonates with prenatal SRI exposure.1 In this study such adverse neonatal outcomes appear to be to some extent moderated by SLC6A4 genotype, suggesting a gene– environment interaction that may account for why some neonates are affected and others are not. The evidence that the SLC6A4 variants moderate the effect of SRI exposure remains ambiguous. Poor neonatal outcomes in this setting are multifactoral and may also reflect a multigenetic process and the gene– environment interaction studied here may be one of many interdependent factors that explain the risk in this setting. In this study infant SLC6A4 genotype appeared to account for some, but not all of the adverse outcomes following prenatal SRI exposure. There are numerous other factors that possibly influence neonatal health in this setting including other transporter regularity related genes, variable number of tandem repeats,39 drug-related metabolism (that is, genotype for CYP450 isoenzymes40), differences in pharmacological properties of the medications themselves41 and other transporter molecules.42 Molecular Psychiatry This study’s sample size was small and further studies are needed to confirm our findings, explore the role of concurrent pharmacological factors and further our understanding of the long-term consequences of reduced 5 min Apgar scores and PNA symptoms in exposed neonates. These findings may help elucidate a component of the mechanisms that underlie PNA and may reflect increased or selective teratogenicity, following prenatal SRI exposure. In turn, these factors contribute to the development of empiric, evidence-based approaches for SRI treatment of persistent maternal mental illness during pregnancy. Pregnancy is a time of highest vulnerability in the reproductive period in a woman’s life for developing complex mental illness and in women with moderate to severe symptoms, pharmacologic treatment may be warranted for stability of maternal mood as depressed mood have predictable long-term consequences for mothers and their children.13,43 Further study is needed to identify the right ‘fit’ between a pharmacological agent and maternal and neonatal genetic factors, which together contribute to successful management of maternal mental illness while minimizing adverse neonatal outcomes. Acknowledgments This research was funded by March of Dimes Foundation (USA) (No. 12-FY01-30), the Canadian Institutes of Health Research (CIHR No. MOP 54490 and 57837) and HELP, UBC (Human Early Learning Partnership). TFO is supported by a HELP, Senior Career Award and has the R Howard Webster Professorship in Child Development (UBC, Faculty of Graduate Studies). CJDR is supported by CIHR and Michael Smith Foundation for Health Research Fellowships. This work was also supported by a grant from the CIHR and Canada Research Chair in Genetics and Behaviour to EMS. We are grateful to the mothers and their infants who participated and contributed to this work, as well as to Colleen Fitzgerald and Ursula Brain for their contributions organizing and facilitating this research program. We also gratefully acknowledge the thoughtful comments provided by Ursula Brain and Tracey Weir and the manuscript reviewers from Molecular Psychiatry. References 1 Moses-Kolko EL, Bogen D, Perel J, Bregar A, Uhl K, Levin B et al. Neonatal signs after late in utero exposure to serotonin reuptake inhibitors: literature review and implications for clinical applications. JAMA 2005; 18, 293 2372–2383. 2 Casper RC, Fleisher BE, Lee-Ancajas JC, Gilles A, Gaylor E, DeBattista A et al. 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