The Impact of Prenatal Alcohol Exposure on Longitudinal Growth, Nutritional Status, and Insulin-Like Growth Factor 1 in Early Childhood in Leyte, the Philippines.

OBJECTIVE: To assess the impact and potential mechanistic pathways of prenatal alcohol exposure (PAE) on longitudinal growth and nutritional status in early childhood. STUDY DESIGN: A cohort of 296 mother-infant dyads (32% with PAE vs 68% unexposed) were recruited in Leyte, the Philippines, and followed from early gestation through 24 months of age. PAE was assessed using serum phosphatidylethanol (PEth) captured twice prenatally and in cord blood and supplemented with self-reported alcohol consumption. Linear mixed models were used to examine longitudinal effects of PAE on growth from birth through 2 years including key potential mediating factors (placental histopathology, and infant serum leptin and Insulin-like Growth Factor 1 [IGF-1]). RESULTS: After adjusting for potential confounders, we found that PAE was significantly associated with a delayed blunting of linear growth trajectories (height-for-age z-score, body length) and weight (weight-for-age z-score, body weight) that manifested between 4 and 6 months and continued through 12-24 months. PAE was also associated with a decreased rate of mid-upper-arm circumference growth from birth to 12 months, and a lower mean IGF-1 levels at birth and 6 months. CONCLUSION: This study demonstrates a delayed impact of PAE on growth that manifested around 6 months of age, underscoring the importance of routine clinical monitoring in early childhood. Furthermore, the findings supported prior animal model findings that suggest a mechanistic role for IGF-1 in PAE-induced growth delay.

Early-life matters: The role of fetal adrenal steroids in the relationship between cytokines within the placental circulation and cognitive development among infants in the Philippines.

Prenatal exposure to inflammation is related to the risk for cognitive impairment in offspring. However, mechanisms underlying the link between inflammatory cytokines at the maternal-fetal interface and human cognitive development are largely unknown. This study addressed this research gap by examining whether i) cytokines within the placenta are associated with different domains of neurocognitive development during infancy, and ii) if DHEA-S in cord blood mediates these associations. We also explored the role of early-life socioeconomic status (SES) in moderating the effect of fetal adrenal steroids on cognitive development in low- and middle-income country contexts. A cohort of 242 mother-infant dyads in Leyte, the Philippines participated in the study and all of them were followed from early pregnancy until 12-months. Concentrations of pro- and anti-inflammatory cytokines in the placenta, and DHEA-S in cord blood collected at delivery were evaluated. The multifactorial aspects of the infant's cognitive functioning were assessed based on the Bayley Scales of Infant Development, third edition (BSID-III). We used Structural Equation Modelling (SEM) with an orthogonal rotation to examine associated paths among latent variables of pro- and anti-inflammatory cytokines in the placenta, fetal neuroendocrine factors, and cognitive development. Pathway analyses showed that both pro- and anti-inflammatory cytokines in the placenta were indirectly related to cognitive (p < 0.05) and language developmental outcomes (p < 0.1) via DHEA-S in cord blood among the low SES group. Yet, we found no statistically significant indirect effect of pro- or anti-inflammatory cytokines on neurocognitive development among the high SES sub-sample. This study extends our understanding of how early-life socioeconomic conditions modify biological pathways underlying the relationship between prenatal factors and postpartum cognitive development.

Inflammatory Myofibroblastic Tumor of the Placenta With Subsequent Successful Pregnancy and Benign Hysterectomy: A Case Report With 59-Month Follow-up.

Inflammatory myofibroblastic tumors (IMT) are rare neoplasms of intermediate malignant potential which have been described in the gynecologic tract, predominantly in the myometrial wall, but also in association with the placenta. Like those in other organs, IMT of the placenta are characterized by molecular abnormalities, most commonly anaplastic lymphoma kinase gene rearrangements, and are often positive for anaplastic lymphoma kinase immunohistochemically. Although the clinical behavior of placental IMTs has so far proven benign, a successful intrauterine pregnancy with subsequent negative hysterectomy following a placental IMT has not been documented. Herein is presented a case of a 27-yr-old noted to have a 2 cm IMT of the extraplacental membranes at delivery, after which the patient received no further treatment. After 56 mo, the patient experienced a subsequent normal delivery in a pregnancy complicated by gestational diabetes. No longer desiring fertility, the patient elected to have a hysterectomy to confirm the absence of IMT at 59 mo and the uterus was unremarkable. This case provides insight into possible outcomes for patients with a rare tumor who may desire future fertility and may otherwise be advised to undergo hysterectomy in the setting of an unclear clinical course.

Placental Findings in Infants with Hypoxic-Ischemic Encephalopathy: The Importance of the Comparison Group.

OBJECTIVE: To determine the effect of 3 distinct comparison groups on associations between placental abnormalities and neonatal hypoxic-ischemic encephalopathy (HIE). STUDY DESIGN: This single-center, prospective case-control study of singletons of gestational age ≥36 weeks with predefined criteria for HIE (n = 30) and 3 control groups was conducted from June 2015 to January 2018. The control groups were infants born by repeat cesarean delivery (n = 60), infants born small for gestational age (SGA; n = 80), and infants receiving positive-pressure ventilation (PPV) at birth (n = 70). One pathologist blinded to infant category reviewed placental sections using the Amsterdam Placental Workshop criteria. Logistic regression with group contrasts relative to HIE was used to analyze primary placental pathologies, and ORs with 95% CIs provided effect sizes. RESULTS: The odds of maternal vascular malperfusion were increased among HIE group placentas compared with placentas of the repeat cesarean delivery (OR, 4.50; 95% CI, 1.45-14.00) and PPV (3.88; 1.35-11.16) groups, but not those of the SGA group. The odds of fetal vascular malperfusion were increased in the HIE group compared with the SGA group (OR, 9.75; 95% CI, 1.85-51.51). The odds of acute chorioamnionitis were higher in the HIE group compared only with the repeat cesarean delivery group, reflecting a similar incidence of chorioamnionitis in SGA group and PPV group placentas. The absence of placental findings was lowest in the HIE group (6.7%), followed by the SGA (18.8%), PPV (31.4%), and repeat cesarean delivery (75%) groups. CONCLUSIONS: Associations with placental abnormalities among infants with HIE varied based on the specific placental abnormality and the control group. Potentially important associations between placental pathology and HIE may be obscured if control groups are not well designed.

Schistosomiasis japonica during pregnancy is associated with elevated endotoxin levels in maternal and placental compartments.

Schistosomiasis affects approximately 40 million women of reproductive age and has been linked to elevated levels of circulating endotoxin in nonpregnant individuals. We have evaluated endotoxin levels in maternal, placental, and newborn blood collected from women residing in Leyte, Philippines. Endotoxin levels in both maternal and placental compartments in pregnant women with schistosomiasis were 1.3- and 2.4-fold higher, respectively, than in uninfected women. In addition, higher concentrations of endotoxin in placental blood were associated with premature birth, acute chorioamnionitis, and elevated proinflammatory cytokines. By promoting endotoxemia, schistosomiasis may exert additional, maladaptive influences on pregnancy outcomes.

Monochorionic twins discordant for mosaic trisomy 14.

Monochorionic-diamniotic twins are usually monozygotic twins and known to be associated with adverse obstetric and perinatal outcomes. Cases of discordant karyotype of monozygotic twins are rare and most involves sex chromosomes. We present the first case of monochorionic twins with discordant karyotype manifested as mosaic trisomy 14 in one twin (B) and a normal karyotype in the other (A). We describe the postmortem pathological and imaging findings of the trisomic twin and for the first time neuropathological findings of this entity. Metaphase chromosome analysis of twin B using fetal tissue showed a 47,XX, +14 karyotype. Chromosomal microarray analysis (CMA) using fetal tissue revealed 38% mosaicism. CMA with placental tissue from both sides demonstrated normal karyotype and confirmed monozygosity, highlighting the value of array based testing on diagnosing mosaicism and zygosity.

Associations between placental pathology and poor intrauterine growth among a cohort of mother-infant singleton pairs in Leyte, the Philippines.

OBJECTIVE: Poor intrauterine growth has negative impacts for child growth and development and disproportionately affects children living in low-resource settings. In the present study, we investigated relationships between placental pathologies and indicators of poor intrauterine growth. METHODS: We enrolled a longitudinal cohort of 279 mother-infant pairs from Leyte, the Philippines. Placental measures included characteristics, pathological findings, and immunohistochemistry. At birth, intrauterine growth was assessed using anthropometric measures, weight-for-gestational age, and the clinical assessment of nutritional status score (CANSCORE) for determining fetal malnutrition. Multivariate linear regression and log-binomial regression models were applied, controlling for potential confounding factors. RESULTS: Maternal vascular malperfusion (MVM) was related to reduced birthweight (P < 0.0001), birth length (P = 0.002), head circumference (P = 0.001), and weight-to-length ratio (P = 0.016). MVM increased the risk for preterm delivery (P = 0.0005) and small for gestational age (SGA) (P = 0.016). Acute chorioamnionitis (P = 0.013) and MVM (P = 0.021) both led to an increased risk for fetal malnutrition defined by CANSORE<25. Villous tissue activated caspase-3 was associated with lower birth length (P = 0.0006), higher weight-to-length ratio (P = 0.004), reduced risks for SGA (P = 0.011) and low weight-to-length ratio for gestational age (P = 0.004). CONCLUSION: The present study applied comprehensive measures for intrauterine growth and demonstrates that low placental weight and placental pathology, chiefly MVM, contribute to poor intrauterine growth. A better understanding of the mechanistic role of specific placental pathologies on adverse newborn outcomes will provide opportunities for reducing incidence of poor intrauterine growth and associated long-term morbidities.

Schistosome egg antigens elicit a proinflammatory response by trophoblast cells of the human placenta.

Schistosomiasis affects nearly 40 million women of reproductive age. Many of these women are infected while pregnant and lactating. Several studies have demonstrated transplacental trafficking of schistosome antigens; however, little is known regarding how these antigens affect the developing fetus and placenta. To evaluate the impact of schistosomiasis on trophoblasts of the human placenta, we isolated primary trophoblast cells from healthy placentas delivered at term. These trophoblasts were placed in culture and treated with Schistosoma japonicum soluble egg antigens (SEA) or plasma from S. japonicum-infected pregnant women. Outcomes measured included cytokine production and activation of signal transduction pathways. Treatment of primary human trophoblast cells with SEA resulted in upregulation of the proinflammatory cytokines interleukin 6 (IL-6) and IL-8 and the chemokine macrophage inflammatory protein 1α (MIP-1α). Cytokine production in response to SEA was dose dependent and reminiscent of production in response to other proinflammatory stimuli, such as Toll-like receptor 2 (TLR2) and TLR4 agonists. In addition, the signaling pathways extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal protein kinase (JNK), p38, and NF-κB were all activated by SEA in primary trophoblasts. These effects appeared to be mediated through both carbohydrate and protein epitopes of SEA. Finally, primary trophoblasts cocultured with plasma from S. japonicum-infected pregnant women produced increased levels of IL-8 compared to trophoblasts cocultured with plasma from uninfected pregnant women. We report here a direct impact of SEA on primary human trophoblast cells, which are critical for many aspects of a healthy pregnancy. Our data indicate that schistosome antigens can activate proinflammatory responses in trophoblasts, which might compromise maternal-fetal health in pregnancies complicated by schistosomiasis.

Dietary soy prevents fetal demise, intrauterine growth restriction, craniofacial dysmorphic features, and impairments in placentation linked to gestational alcohol exposure: Pivotal role of insulin and insulin-like growth factor signaling networks.

INTRODUCTION: Prenatal alcohol exposure can impair placentation and cause intrauterine growth restriction (IUGR), fetal demise, and fetal alcohol spectrum disorder (FASD). Previous studies showed that ethanol's inhibition of placental insulin and insulin-like growth factor, type 1 (IGF-1) signaling compromises trophoblastic cell motility and maternal vascular transformation at the implantation site. Since soy isolate supports insulin responsiveness, we hypothesized that dietary soy could be used to normalize placentation and fetal growth in an experimental model of FASD. METHODS: Pregnant Long-Evans rat dams were fed with isocaloric liquid diets containing 0% or 8.2% ethanol (v/v) from gestation day (GD) 6. Dietary protein sources were either 100% soy isolate or 100% casein (standard). Gestational sacs were harvested on GD19 to evaluate fetal resorption, fetal growth parameters, and placental morphology. Placental insulin/IGF-1 signaling through Akt pathways was assessed using commercial bead-based multiplex enzyme-linked immunosorbent assays. RESULTS: Dietary soy markedly reduced or prevented the ethanol-associated fetal loss, IUGR, FASD dysmorphic features, and impairments in placentation/maturation. Furthermore, ethanol's inhibitory effects on the placental glycogen cell population at the junctional zone, invasive trophoblast populations at the implantation site, maternal vascular transformation, and signaling through the insulin and IGF1 receptors, Akt and PRAS40 were largely abrogated by co-administration of soy. CONCLUSION: Dietary soy may provide an economically feasible and accessible means of reducing adverse pregnancy outcomes linked to gestational ethanol exposure.

Fetal Ovarian Reserve: the Dynamic Changes in Ubiquitin C-Terminal Hydrolase L1.

The regulation of protein turnover by the unique deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCHL1) is only seen in oocytes, spermatogonia, and neurons. Our objective was to investigate variation in expression of UCHL1 across fetal maturation of oocytes that result in lifelong ovarian reserve. We performed a retrospective cohort study of 25 fetal autopsy specimens from 21 to 36 weeks. This was an IRB-approved protocol with parental permission for use of tissues for research purposes. Tissues were stained for expression of the oocyte-specific protein UCHL1, and expression levels were evaluated using quantitative immunofluorescence across gestational ages after correction for the area and background absorbance. Corrected total cell fluorescence (CTCF) for expression of UCHL1 within human oocytes was compared across fetal gestational ages and oocyte size. Trends were analyzed using a locally weighted scatterplot smoothing algorithm. Local expression of UCHL1 increases in oocytes across ovarian development reaching a plateau at 27 weeks with the maintenance of elevated levels through 36 weeks gestational age. This maturation trend is also evidenced by the increase in protein expression as oocyte area increases (r = 0.5530, p ≤ 0.001) with the largest rise occurring as oocytes are enveloped into primordial follicles. The increase in expression as oocytes transition from oogonia into oocytes in primordial follicles and beyond may be part of the preparation of both oocytes and the surrounding somatic cells for the long-term maintenance of the ovarian reserve.

Maternal Schistosomiasis japonica is associated with maternal, placental, and fetal inflammation.

Schistosomes infect ∼40 million women of childbearing age and result in the elaboration of proinflammatory cytokines that have been implicated in fetal growth restriction. In murine models and two observational studies in humans, schistosome infection during pregnancy was associated with reduced birth weight, although a recent treatment trial in Schistosoma mansoni did not detect this association. We conducted an observational study among 99 pregnant women living in an area of Schistosoma japonicum endemicity in the Philippines. We enrolled women at 32 weeks gestation and measured S. japonicum and geohelminth infection intensity. We collected maternal peripheral blood at 32 weeks gestation and placental and cord blood at delivery to assess inflammatory status. At delivery, we collected a placental-tissue sample and measured birth weight. In multivariate models adjusted for geohelminths, maternal schistosomiasis was associated with increased levels of inflammatory cytokines in maternal peripheral (tumor necrosis factor alpha [TNF-α] and interleukin 10 [IL-10]), placental (TNF-α, IL-6, TNF-α receptor II [RII], and IL-1ß), and cord (IL-1ß and TNF-α RII) blood, as well as acute subchorionitis and increased TNF-α production by syncytiotrophoblasts assessed by immunohistochemistry (all P < 0.05). After adjusting for confounders, placental IL-1ß, and TNF-α production by syncytiotrophoblasts was independently associated with decreased birth weight (both P < 0.05). Our data indicate that maternal schistosomiasis results in a proinflammatory signature that is detectable in maternal, placental, and fetal compartments, and a subset of these responses are associated with decreased birth weight. This potential mechanistic link between maternal schistosomiasis and poor birth outcomes will contribute to the debate regarding treatment of maternal schistosome infections.

Ethanol-induced oxidative stress and mitochondrial dysfunction in rat placenta: relevance to pregnancy loss.

BACKGROUND: Ethanol consumption during pregnancy increases the risk of early pregnancy loss and causes intrauterine growth restriction. We previously showed that chronic gestational exposure to ethanol impairs placentation, and that this effect is associated with inhibition of insulin and insulin growth factor signaling. Since ethanol also causes oxidative stress and DNA damage, we extended our investigations to assess the role of these pathological processes on placentation and placental gene expression. METHODS: Pregnant Long Evans rats were pair-fed liquid diets containing 0% or 24% ethanol by caloric content. Placentas harvested on gestation day 16 were used to examine DNA damage, lipid peroxidation, apoptosis, mitochondrial gene/protein and hormonal gene expression in relation to ethanol exposure. RESULTS: Gestational exposure to ethanol increased fetal resorption, and trophoblast apoptosis/necrosis, oxidative stress, DNA damage, and lipid peroxidation. These adverse effects of ethanol were associated with increased expression of pro-apoptotic (Bax and Bak) and reduced levels of the anti-apoptotic Bcl-2 protein. In addition, increased trophoblast apoptosis proneness was associated with p53-independent activation of p21, reduced mitochondrial gene and protein expression, and dysregulated expression of prolactin (PRL) family hormones that are required for implantation and pregnancy-related adaptations. CONCLUSIONS: Chronic gestational exposure to ethanol increases fetal demise due to impaired survival and mitochondrial function, increased oxidative stress, DNA damage and lipid peroxidation, and dysregulated expression of prolactin family hormones in placental trophoblasts.

Placental findings among newborns with hypoxic ischemic encephalopathy.

OBJECTIVE: To determine if pre-specified placental abnormalities among newborns with hypoxic-ischemic encephalopathy (HIE) differ compared to newborns admitted to a NICU without encephalopathy. STUDY DESIGN: Retrospective case-control study of newborns with HIE (2006-2014) and controls matched for birth year, gestational age, weight, and gender. One pathologist reviewed archived placental sections using pre-specified criteria. RESULTS: Sixty-seven newborns had HIE, 46 had available placentas and were matched with 92 controls. HIE had more maternal vascular malperfusion (46% vs 25%, p = 0.02), fetal vascular malperfusion (13% vs 0%, p < 0.001), and clinical abruption (22% vs 4%, p = 0.001). Controls had more normal placentas (29% vs 7%, p = 0.002), and chorioamnionitis (30% vs 9%, p = 0.005). Pre-specified placental lesions occurred in 87% of HIE and 65% of controls (p = 0.008) and identified >90% of primary diagnoses. CONCLUSIONS: Pre-specified placental lesions identified nearly all abnormalities in HIE and represented both acute and chronic processes.

Role of aspartyl-(asparaginyl) beta-hydroxylase in placental implantation: Relevance to early pregnancy loss.

Aspartyl-(asparaginyl) beta-hydroxylase (AAH) is a type 2 transmembrane protein with catalytic activity that hydroxylates epidermal growth factor-like domains of proteins that have a functional role in cell motility and invasion. Extravillous cytotrophoblasts (CTB) are motile and invasive unpolarized epithelial cells that mediate early implantation through interaction with the endometrium. This study characterizes the potential role of AAH in CTB implantation using human placentas from (1) terminated pregnancies (n = 11), (2) normal term deliveries (n = 21), (3) spontaneous abortuses (n = 21), and (4) small-for-gestational-age (SGA) term deliveries (n = 21). The SGA cases all had established clinical histories of intrauterine growth restriction or preeclampsia. Formalin-fixed, paraffin-embedded sections of placenta were immunostained using the 15C7 monoclonal antibody generated to recombinant AAH. In addition, snap-frozen or RNAlater-preserved specimens (Ambion, Austin, TX) were used for RNA analysis of AAH expression by real-time quantitative reverse transcriptase-polymerase chain reaction and protein analysis by Western blotting. The immunohistochemical staining studies demonstrated AAH expression in amniocytes, villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands at all gestational ages and in all 4 groups. Higher levels of AAH immunoreactivity were observed in extravillous CTB compared with villous CTB. Immunohistochemical staining and RNA analysis demonstrated abundant AAH expression in placental trophoblastic cells as well as in decidua and endometrial glands, with reduced expression in spontaneous abortion and SGA, suggesting that AAH may serve as a biomarker of impaired implantation. The high levels of AAH in decidua and endometrial glands suggest a role for this molecule in "receptivity" of endometrium.

Redness discordance in monochorionic twin placentas: Correlation with clinical and placental findings.

INTRODUCTION/OBJECTIVES: Recent studies suggest redness (color) discordance of the placental basal plate may be a marker for twin anemia-polycythemia sequence (TAPS), a recently described complication of diamniotic-monochorionic twinning characterized by marked intertwin hemoglobin (Hb) discordance in the absence of oligohydramnios-polyhydramnios. In this study, we determined the clinicoplacental and choriovascular correlates of basal plate color discordance in monochorionic twin placentas, and assessed its value as postnatal indicator of TAPS. METHODS: We performed a clinicoplacental analysis of 100 consecutive non-TTTS diamniotic-monochorionic twin placentas with available photographic documentation of the basal plate. Basal plate redness was quantified by computer-assisted analysis of digital images and expressed as intertwin color difference ratio (CDR). RESULTS: The CDR ranged between 1.00 and 3.58 (median CDR: 1.14; 90th %ile: 1.98). Compared to twins with low CDR (N = 90), twins with high CDR (≥2.0; N = 10) had significantly higher hemoglobin difference (11.25 g/dL versus 2.55 g/dL) and significantly fewer and smaller artery-to-artery (AA) and artery-to-vein (AV) anastomoses. Apgar scores and birth weights were equivalent in both groups. Among the 10 twin sets with high CDR, six (60%) qualified as TAPS, as defined by intertwin Hb difference >8 g/dL and absent or very small AA and AV anastomoses. Conversely, 6 of 8 (75%) twin sets with TAPS had a CDR ≥ 2.0. CONCLUSION: Intertwin CDR correlates with intertwin hemoglobin difference and chorionic angioarchitecture. A CDR value ≥ 2.0 (the 90%ile value for CDR derived from the present cohort) has high specificity (96%), but relatively low positive predictive value (60%) as indicator of TAPS, as currently defined.

Potential Contributions of the Tobacco Nicotine-Derived Nitrosamine Ketone to White Matter Molecular Pathology in Fetal Alcohol Spectrum Disorder.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is associated with long-term deficits in cognitive and motor functions. Previous studies linked neurodevelopmental abnormalities to increased oxidative stress and white matter hypotrophy. However, similar effects occur with low-dose nitrosamine exposures, alcohol abuse correlates with cigarette smoking, and tobacco smoke contains tobacco-specific nitrosamines, including NNK. HYPOTHESIS: Tobacco smoke exposure is a co-factor in FASD. DESIGN: Long Evans rat pups were i.p. administered ethanol (2 g/kg) on postnatal days (P) 2, 4, 6 and/or NNK (2 mg/kg) on P3, P5, and P7 to simulate third trimester human exposures. Oligodendroglial myelin-associated, neuroglial, and relevant transcription factor mRNA transcripts were measured using targeted PCR arrays. RESULTS: Ethanol and NNK differentially altered the expression of immature and mature oligodendroglial, neuronal and astrocytic structural and plasticity-associated, and various transcription factor genes. NNK's effects were broader and more pronounced than ethanol's, and additive or synergistic effects of dual exposures impacted expression of all four categories of genes investigated. CONCLUSION: Developmental exposures to alcohol and NNK (via tobacco smoke) contribute to sustained abnormalities in brain white matter structure and function via distinct but overlapping alterations in the expression of genes that regulate oligodendrocyte survival, maturation and function, neuroglial structural integrity, and synaptic plasticity. The results support the hypothesis that smoking may contribute to brain abnormalities associated with FASD.

Differential Effects of 3rd Trimester-Equivalent Binge Ethanol and Tobacco-Specific Nitrosamine Ketone Exposures on Brain Insulin Signaling in Adolescence.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is associated with impairments in insulin and insulin-like growth factor (IGF) signaling through Akt pathways and altered expression of neuro-glial proteins needed for structural and functional integrity of the brain. However, alcohol abuse correlates with smoking, and tobacco smoke contains 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which like other nitrosamines, impairs insulin and IGF signaling. HYPOTHESIS: NNK exposure can serve as a co-factor in mediating long-term neuro-developmental abnormalities associated with FASD. DESIGN: Long Evans rat pups were IP administered ethanol (2 g/kg) on postnatal days (P) 2, 4, 6 and/or NNK (2 mg/kg) on P3, P5, and P7, simulating third trimester human exposures. Temporal lobes from P30 rats (young adolescent) were used to measure signaling through the insulin/IGF-1/Akt pathways by multiplex ELISAs, and expression of neuroglial proteins by duplex ELISAs. RESULTS: Ethanol, NNK, and ethanol + NNK exposures significantly inhibited insulin receptor tyrosine phosphorylation, and IRS-1 and myelin-associated glycoprotein expression. However, the major long-term adverse effects on Akt pathway downstream signaling and its targeted proteins including choline acetyltransferase, Tau, pTau, ubiquitin, and aspartate-ß-hydroxylase were due to NNK rather than ethanol. CONCLUSION: Alcohol and tobacco exposures can both contribute to long-term brain abnormalities currently regarded fetal ethanol effects. However, the findings suggest that many of the adverse effects on brain function are attributable to smoking, including impairments in signaling through survival and metabolic pathways, and altered expression of genes that regulate myelin synthesis, maturation and integrity and synaptic plasticity. Therefore, public health measures should address both substances of abuse to prevent "FASD".