There is growing interest in the molecular mechanisms which underlie the epidemiologic observations of the developmental origins of health and disease, as well as a focus of study on the critical role of the placenta in mediating these effects[16
]. Most studies to date have emphasized gross placental morphology and its relationship to near-term adverse birth outcomes or later life cardiovascular disease and metabolic syndrome related phenotypes[28
]. We systematically quantify imprinted gene expression using a targeted qRT-PCR strategy in the placenta and to relate variability in that expression to another important phenotype, infant neurobehavioral profiles. These results support a critical role for the function of the placenta and for genomic imprinting of genes in the placenta in neurodevelopmental outcome. The neurobehavioral profiles captured through the NICU Network Neurobehavioral Scales assessment have robust, prospective predictive potential for later behavior problems, school readiness, and IQ through 4.5 years of age[21
]. Thus the associations we have identified between the placenta imprinted gene expression profiles and NICU Network Neurobehavioral Scales informs the molecular mechanism through which the intrauterine environment plays a role in mental health and behavior beyond birth.
Variability in expression of imprinted genes in this population is consistent with our previous work, where we observed up to 2–4 fold variation in expression in human placentas[23
]. This would indicate that mechanisms other than classical loss of imprinting may be affecting gene expression, as loss of imprinting has a maximum effect of doubling the expression level. Our observation of a strong correlation in the expression between many of the imprinted genes, and most particularly amongst genes located on the same chromosomal regions suggest coordinated alterations in expression potentially results from a common stress or exposure encountered during intrauterine development and/or disruption of a single imprinting control region. This also suggests that the association of complicated phenotypes, such as neurobehavioral profiles, with changes in gene expression may not result from a single altered gene, but from coordinated alterations in a number of genes. Multiple gene expression signatures are now being recognized as important predictors of outcome in oncologic diagnostics[32
]. This observation should extend to other complex outcomes and diseases. Our statistical modeling allows for detailed but unbiased examination of imprinted gene expression as expression profiles, thereby improving our power and taking into account this potentially important coordinated variation.
The placenta plays a key role in regulating the intrauterine environment and in the appropriate development of the fetus. Once developed, the placenta serves as the source of fetal nutrients, water and gas exchange, excretion of toxic waste products, and serves many immune-endocrine interactions at the maternal fetal interface. These effects are modulated by simultaneous production of many pregnancy related hormones, proteins and growth factors thereby fulfilling a critical role in proper intrauterine development. The placenta produces an array of neuropeptide hormones that are analogous to those produced by the hypothalamus and pituitary of the brain, including GnRH, TRH, GHRH, CRH, and oxytocin[33
]. Rapid advancements in understanding the integrated regulation of neuropeptide/neuroendocrine homeostasis within and outside the brain as well as placenta[34
] has led to the formulation of a new concept that placenta is the “third brain” that links the developed (maternal) and developing (fetal) brains [35
], and have expanded the role of the placenta in the pathophysiology of intrauterine insults[16
]. Thus, altered placental function, through altered expression of imprinted genes may play a critical role in mediating various health outcomes related to the intrauterine environment[37
We identified that infants’ whose placentas exhibited an expression profile of class 4 had significantly reduced quality of movement in the NNNS assessment. There were a number of infants in this category who exhibited scores considered in the lowest 10th
percentile from normative scales of performance[22
]. These measures examine motor control including smoothness, maturity, and lack of startles and tremors. Reduced quality of movement has been observed among infants exposed to prenatal methamphetamine[27
], infants born small for gestational age infants to adolescent mothers[38
], and infants requiring pharmaceutical treatment for neonatal abstinence syndrome[39
]. Our findings, in non-drug exposed infants suggest that there may be other exposures or stressors during the intrauterine developmental period that can, through alterations in the expression of imprinted genes, and thus the function of the placenta, lead to similar poorer quality of movement phenotypes. Imprinted genes such as MEG3
, and HOXD10
demonstrated greatly reduced expression in infants with this expression profile compared with those in class 1, and network analysis suggests these genes have functional overlap in nervous system, skeletal, and muscular development. HOXA11
plays a key role in joint and skeletal development[40
is important in motorneuron development[41
], and MEG3
alterations have been linked with Prader-Willi syndrome like phenotypes[42
]. These genes encode non-coding RNAs, which may play a critical role in modifying placental gene expression, and may have resulted in the observed phenotypes.
Both classes 2 and 4 showed greater handling scores, suggesting more handling strategies were needed to maintain the infant in an alert state during the NICU Network Neurobehavioral Scales orientation examination. The observed relationship between handling score and imprinted gene expression class was most similar for the genes CDKAL1
, and PHLDA2. ILK
is highly expressed in first trimester placentas, regulating migration, and signaling through ILK may be involved in trophoblast syncytialization[43
is related to placental angiogenesis[45
], and increased expression of placental maternally expressed PHLDA2
has been associated with reduced infant birthweight[46
]. An Ingenuity network analysis suggested that these genes are involved in cell cycle control and cell growth, and it may be possible that it is through these functions and their role in placental development that these genes can influence neurodevelopment.
A limitation of this study is the moderate sample size in examining the large number of neurobehavioral outcomes available through the NICU Network Neurobehavioral Scales assessment. The length of follow-up is also limited for these infants, so definitive relationships between imprinting signatures and long-term neurobehavioral outcomes await validation. Finally, our samples are limited to term placentas, which may not accurately reflect the expression pattern throughout pregnancy. We cannot make definitive statements regarding the mechanistic link between these profiles, neurobehavior, and birth at earlier gestational ages. Nonetheless, we evaluated a representative collection of samples from the single, large, high-risk delivery service which serves an entire geographic region.
In summary, this study provides support for relationships between imprinted gene expression and critical, prospectively predictive early neurobehavioral measures. Replication and expansion of these findings in additional cohorts is warranted, with additional examination of the important confounding clinical factors during pregnancy which mediate the variation in imprinted gene expression and the relationship to developmental outcome. This research also raises critical questions on the mechanisms through which imprinted genes become deregulated, if and for how long their alteration may persist beyond birth, and the potential for additional neurodevelopmental and other health outcomes to be associated with this altered expression.