J.A. Russell et al. (Eds.) Progress in Brain Research, Vol. 133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 9

The neurobiology of stress in human pregnancy: implications for prematurity and development of the

fetal central nervous system

Pathik D. Wadhwa 1,2,,, Curt A. Sandman 1 and Thomas J. Garite 2

! Department of Psychiat~ and Human Behavior, University of California, Irvine, College of Medicine, 3117 Gillespie Neuroscience Building, lrvine, CA 92697, USA

2 Department of Obstetrics and Gynecology, University of California, Irvine, College of Medicine, 3117 Gille~pie Neuroscience Building, lrvine, CA 92697, USA

Abstract: Adverse early experience, including prenatal maternal psychosocial stress, has the potential to negatively influence developmental processes through both physiological and behavioral mechanisms. This in turn may have adverse consequences for the mental and physical health, well-being and aging of the individual throughout the entire life-span. We have initiated a program of research on humans to examine the consequences of maternal stress and related factors in pregnancy on the length of gestation, fetal growth, and brain development. We have also investigated the physiological mechanisms that are involved. In this chapter we outline the theoretical rationale for this work and give an overview of our findings to date. These findings support a significant and independent role for behavioral processes such as maternal prenatal stress in the etiology of prematurity-related outcomes, and suggest that these effects are mediated, in part, by the maternal-placental-fetal neuroendocrine axis; specifically by placental corticotropin-releasing hormone. Using a fetal challenge paradigm as a novel method for quantifying fetal neurologic maturity in utero, we have found that the maternal environment exerts a significant influence on the fetal autonomic nervous system and on central nervous system processes related to recognition, memory and habituation. Finally, our findings provide preliminary evidence to support the notion that the influence of prenatal stress and maternal-placental hormones on the developing fetus may persist after birth, as assessed by measures of temperament and behavioral reactivity in the first 3 years of postnatal life. The implications of these studies for life-span development and health are discussed.

Introduction

Developmental processes involved in transforming a single-cell human embryo into a fully functioning organism whose cerebral cortex alone contains some 109 neurons, within a mere span of 40 weeks, are exceedingly complex and fascinating. It would be

* Corresponding author: Pathik D. Wadhwa, Behavioral Perinatology Research Program, University of California, Irvine, 3117 Gillespie Neuroscience Building, Zot Code 4260 Irvine, CA 92697, USA. Tel.: +1-949-824-8238; Fax: +1-949-824-8218; E-mail: pwadhwa@uci.edu

difficult to find another example in the physical or biological world that even begins to approximate the elegance of intrauterine development. The develop- ment of the fetus is characterized by proliferation and specialization of cells to form interconnected functional units. Whereas most of the structural and functional development of major organ systems is essentially complete by the end of the first trimester of gestation, the central nervous system (CNS) con- tinues to grow and to develop throughout gestation and for the first 10-12 years of life after birth. CNS development may be characterized into various stages, overlapping in time. These include the prolif- eration and migration of neurons and glial cells, the

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differentiation and maturation of neurons including axonal and dendritic development and synaptogene- sis, myelination, and the development of neurotrans- mitter systems (noradrenergic, dopaminergic, sero- tonergic, cholinergic, y-aminobutyric acid (GABA), excitatory amino acid and peptidergic). During peak growth periods, neurons are generated at the rate of over 250,000 per minute, and by birth the brain con- tains over one hundred trillion connections among neurons (Cowan, 1979; Rakic, 1988; Institute of Medicine, 1992).

Throughout the ages biologists have questioned whether the genetic material of the fertilized egg al- ready contains a full set of building specifications for the human organism? Over the last few years, there has been a major paradigm-shift in developmental neuroscience regarding fundamental concepts of de- velopment and function. The answer to the above question is now believed to be an unequivocal 'no'. Genes and environment are no longer considered to exert separate influences, and development is viewed not as a gradual elaboration of an architectural plan pre-configured in the genes, but rather as a dynamic interdependency of genes and environment. This is characterized by a continuous process of interaction between these two factors in a place- and time- specific manner, and involves short- and long-term information storage, whereby genetic and epigenetic processes become represented in the evolving struc- tural and functional design of the organism at ev- ery step of development (Hofer, 1988; Institute of Medicine, 1992; Smotherman and Robinson, 1995). According to this epigenetic view of development, events at one point in time have consequences that are manifested later in the developmental process, and the outcome of the interaction between genes and environment at any point in development has a profound influence on the developmental trajectory (Kolb, 1995). In other words, it appears that within the constraints imposed by the heritable germ line at conception, each developing organism plays an active role in its own construction. This dynamic process is effected by the emergence of various sys- tems during embryonic and fetal life that acquire information about the nature of the environment, and use this information to guide development. In the context of this formulation, not only does environ- ment play a necessary role for development to occur,

but the nature of the environment may either play an advantageous role that promotes normal or optimal development, or may play a pernicious role which harms development (Bornstein, 1989).

Clearly, if the nature of the environment is per- ceived to be stressful or hostile, it may promote developmental processes that result in deleterious short- and/or long-term consequences for health. For example, using animal models, several experimental studies have provided powerful evidence to support a causal role for prenatal stress in negatively influ- encing critical developmental and health outcomes. These negative effects are upon brain morphology and function (cognition, emotionality, and behav- ior), sexual differentiation, activity of the autonomic nervous, neuroendocrine, immune and reproductive systems, physical health, the time course of normal aging (rate of neuronal loss and cognitive decline), and longevity (Lauder et al., 1981; Fride and Wein- stock, 1984, 1988; Peters, 1986, 1988, 1990; Rohde et al., 1989; Insel et al., 1990; Alonso et al., 1991; Ohkawa et al., 1991; Schneider et al., 1992; Sobrian et al., 1992; Takahashi et al., 1992; Schneider and Coe, 1993; Henry et al., 1994; Uno et al., 1994; Bakker et al., 1995; Barbazanges et al., 1996; Coe et al., 1996; Sanchez et al., 1996). These studies have offered valuable insights into putative physiological mechanisms that may be involved in mediating the effects of stressful environments on the developing organism.

The ability to generalize these findings from an- imals to humans may be limited by the existence of inter-species differences in physiology, however. No single system exemplifies the magnitude of these inter-species differences as vividly as the reproduc- tive system, even when compared between other- wise very closely related species such as humans and non-human primates (Smith, 1999). Our group has been working for the last few years on a pro- gram of research to examine the interface between biology and behavior in the context of human preg- nancy and fetal development. Specifically, we have been interested in evaluating the effects of mater- nal stress during pregnancy on outcomes related to the length of gestation, fetal growth, and fetal brain development. We have begun to examine pu- tative physiological mechanisms that may mediate the effects of the maternal environment on the devel-

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oping fetus, with a particular emphasis on the role of the maternal-placental-fetal neuroendocrine axis. From this work we have articulated a neurobiolog- ical model of prenatal stress, which proposes that stress exerts a significant and negative influence on fetal developmental outcomes, in part by its effects on placental corticotropin-releasing hormone (CRH) production. This model, which is based on an un- derstanding of the ontogeny of fetal development and the endocrinology of human pregnancy, further hypothesizes that the effects of prenatal stress are outcome-specific, and that they are modulated by the nature, timing and duration of stress (Wadhwa, 1998).

The adoption of an epigenetic framework for early development, wherein the organism plays an active role in its own construction by evolving systems to acquire and use information about the nature of the environment to guide development, gives rise to two important questions. Firstly, how do the fetal and ma- ternal compartments communicate with one another? Secondly, in light of the fact that the fetal nervous system is itself in a state of evolution and has yet to acquire its repertoire of structural and functional capabilities, what are the modalities available to the developing fetus to receive, process and act upon information acquired from the environment? There are no direct neural or vascular connections between the mother and her developing fetus, and bi-direc- tional communication is mediated primarily via the exchange of blood-borne chemical signals produced by the endocrine and immune systems, for example (Meschia, 1994). One of the remarkable adaptations of pregnancy is the formation in early gestation of a transient organ of fetal origin - - the placenta. In addition to the long-recognized roles played by the placenta, this organ may also undertake functions that are usually ascribed to the central nervous sys- tem, i.e. the capability of receiving, processing and responding to certain classes of external stimuli. In- deed, we believe that a major role of the placenta is to act on behalf of the fetus as both a sensory and effector organ to facilitate the transduction of environmental information for its incorporation into the developmental process.

The physiology of placental CRH illustrates this concept. This 41-amino acid neuropeptide is of pre- dominantly hypothalamic origin and is one of the pri-

mary mediators through which the brain regulates the activity of the hypothalamo-pituitary-adrenal (HPA) axis and the physiological responses to stress and inflammation (Vale et al., 1981; Chrousos, 1992). However, during human pregnancy, the CRH gene and receptors are also richly expressed in the pla- centa, and this placental CRH is identical to hy- pothalamic CRH in structure, immunoreactivity, and bioactivity (Petraglia et al., 1996). The expression of CRH mRNA increases exponentially over the second half of gestation, effecting the production of CRH in the placenta and its release into the maternal and fetal compartments. We and others have proposed various crucial roles for placental CRH in regulating human pregnancy biology, including modulation of maternal and fetal pituitary-adrenal function, participation in fetal cellular differentiation, growth and maturation, and involvement in the physiology of parturition (Challis et al., 1995; Petraglia et al., 1996). Several convergent lines of evidence suggest that the activity of placental CRH is, in turn, regulated by character- istics of the maternal and intrauterine environment. For example, in vitro and in vivo studies have shown placental CRH output to be modulated in a pos- itive, dose-related manner by the major biological effectors of stress responses, including cortisol, cat- echolamines, oxytocin, angiotensin-II, both forms of interleukin-1, and hypoxia (Petraglia et al., 1987, 1989, 1990; Korebrits et al., 1998a; Marinoni et al., 1998). Therefore we propose that the placenta plays an important role on behalf of the fetus as a sen- sory and effector organ, and more specifically that placental CRH may mediate the effects of the ma- ternal environment on fetal development and related outcomes.

General methodology

Components of our neurobiological model of prena- tal stress have been tested in prospective, longitudi- nal population-based cohort studies with a combined sample of approximately 375 women with singleton, intrauterine pregnancies, recruited during the second trimester of gestation and followed through delivery into the early postpartum period. Our recruitment strategy ensured heterogeneity in terms of sociode- mographic and ethnic characteristics. Based on con- ventional measures of obstetric risk we included

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approximately equal numbers of subjects at low- and high-risk for adverse perinatal outcomes. Structured interviews and questionnaires were administered at two or more time points in the second and third trimesters of gestation using standardized and val- idated instruments to assess maternal psychosocial constructs. These included various forms of prenatal stress, social support, personality, and several mater- nal behaviors, including diet and nutrition, physical activity, and smoking, alcohol and drug use. Ma- ternal venous and umbilical cord blood (plasma) samples were collected during gestation and at deliv- ery for bioassays of stress hormones, including CRH, adrenocorticotropin (ACTH), ~-endorphin, and cor- tisol. Obstetric and birth outcomes were abstracted from the medical record. All pregnancies were dated by best obstetric estimate using last menstrual period and early ultrasonographic confirmation. In a sub- sample of 156 pregnancies, fetal assessments were performed early in the third trimester of gestation. These assessments included fetal biometry, Doppler flow velocimetry of the uteroplacental circulation, and an experimental challenge paradigm to quantify indices of fetal arousal, reactivity, learning and habit- uation, assessed by fetal heart rate (FHR) responses to a series of vibroacoustic stimuli.

Results

Maternal psychosocial processes and birth outcomes

To date, we have completed three studies that have each assessed and provided evidence to support the notion that maternal psychosocial processes signif- icantly predict pregnancy outcomes related to the length of gestation and fetal growth, even after con- trolling for the effects of established sociodemo- graphic and obstetric risk factors.

In the first study, a sample of 90 pregnant women were prospectively assessed using measures of episodic and chronic stress, strain (response to stress), and pregnancy-related anxiety. Results, ana- lyzed using multiple regression analyses, indicated that independent of obstetric risk, each unit increase of prenatal life event stress (from a possible sample range of 14.7 units of life event stress) was signifi- cantly associated with a 55 g decrease in infant birth weight and with a 32% increase in the relative risk of

low birth weight (

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the pattern of physiological responses to exogenous stimuli or perturbations such as stress (Monga and Creasy, 1994; Wadhwa et al., 1996). Therefore we have carried out two studies to examine the nature of the relationship between matemal psychosocial processes and this neuroendocrine system during pregnancy. Results from both studies have supported our hypothesis that the maternal psychosocial en- vironment during pregnancy impacts upon maternal physiology, as measured by the functioning of the maternal pituitary-adrenal axis.

The first study, conducted in a sample of 54 women with a singleton, intrauterine pregnancy, examined the cross-sectional relationships between maternal measures of prenatal stress and social support and maternal plasma concentrations of pituitary-adrenal hormones (ACTH, [3-endorphin, cortisol) at 28 weeks' gestation. Factors known to influence neuropeptide and glucocorticoid hor- mone levels during pregnancy, including gestational age, diurnal rhythm and obstetric risk, were con- trolled for. Our results indicated that maternal stress was positively associated with maternal circulating ACTH and cortisol levels in addition to the preg- nancy-associated elevations in baseline concentra- tions of maternal pituitary-adrenal hormones. In contrast, social support was negatively associated with the circulating concentrations of these hor- mones. Step-wise hierarchical multiple regression analyses, performed to examine the joint contri- bution of prenatal demographic and psychosocial factors to the maternal neuroendocrine parameters, indicated that our predictors accounted for 36% of the variance in maternal ACTH and 13% of the variance in maternal cortisol levels (Wadhwa et al., 1996).

The second study involved administration of a standard, laboratory-based behavioral stressor - - a speech or mathematics task - - to a sample of 22 healthy, normotensive women (15 pregnant women ranging between 12 and 35 weeks' gestation, and 7 non-pregnant controls). This was used to determine whether mild behavioral stress could evoke a reliable physiological response during pregnancy, to com- pare the magnitude of responses between pregnant and non-pregnant women, and to examine whether gestational age at testing and baseline physiologi- cal states predicted the magnitude of physiological

stress reactivity in pregnancy. Sympathetic-adrenal- medullary (SAM) responses were assessed in all sub- jects by quantifying changes in systolic and diastolic blood pressure and heart rate upon application of be- havioral stress. Baseline HPA axis measures (plasma concentrations of ACTH and cortisol) were also ob- tained in all pregnant women. The possible effects of other factors on biological stress reactivity (i.e. high-risk obstetric conditions, endocrine disorders, circadian rhythms) were controlled for by the study design. The results indicated that each of the be- havioral stressors elicited reliable SAM responses in both study groups. The magnitude of these responses was normally distributed across subjects, with suf- ficient variability for individual-difference analyses. SAM responsivity was significantly attenuated in pregnant subjects compared with non-pregnant con- trols. Moreover, among pregnant women there was a progressive increase in attenuation of the SAM response with advancing gestational age, accounted for, in part, by baseline levels of ACTH and cortisol (Wadhwa et al., 1997b).

Maternal pituitary-adrenal function and placental CRH in human pregnancy

To clarify the role of maternal pituitary-adrenal function in modulating placental CRH activity in vivo, we conducted a study in a sample of 260 adult women with a singleton, intrauterine pregnancy by examining the associations between maternal plasma CRH, ACTH, ~-endorphin and cortisol concentra- tions early in the third trimester of pregnancy (31-32 weeks' gestation). A second maternal plasma sample was obtained in a sub-sample of 158 women at 6 weeks postpartum. The results suggested that mater- nal ACTH and [3-endorphin levels during pregnancy and postpartum were highly correlated (P < 0.001), indicating their origin from a common precursor- pro-opiomelanocortin. During pregnancy, CRH lev- els were significantly and positively correlated with ACTH (P < 0.01) and [3-endorphin (P < 0.001) levels. There was no direct association between CRH and cortisol in the present sample of subjects, how- ever both plasma ACTH and [3-endorphin concen- trations were significantly and positively correlated with cortisol concentration (P < 0.001). The time of day of blood sampling was significantly and nega-

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tively correlated with maternal cortisol levels during pregnancy with higher levels in the morning and lower levels in the evening (r = -0.22, P < 0.001), indicating the presence of an intact circadian rhythm for cortisol. However, an analysis of covariance ap- proach to control for the effects of time of day revealed no changes in the magnitude or signifi- cance levels of the above relationships. In contrast to the findings during pregnancy, maternal plasma CRH levels were not related with either ACTH, ~-endorphin, or cortisol during the post-partum pe- riod. Thus, these findings support the premise that in human pregnancy placental CRH activity is modu- lated, in part, in a positive manner by the maternal pituitary-adrenal axis (Wadhwa et al., 1997a).

Placental CRH and prematurity

Endocrine and paracrine/autocrine roles have been proposed for placental CRH in the physiology of parturition and also in fetal growth and develop- ment. We have begun to explore these possibilities by investigating the prospective relationship between concentrations of CRH (derived from the placenta) in maternal plasma early in the third trimester of ges- tation and the risk for prematurity-related outcomes.

This study involved a sample of 63 women with a singleton, intrauterine pregnancy. Maternal plasma was collected at 28-30 weeks' gestation, and CRH concentrations were determined by radioimmunoas- say. The results indicated that maternal (placental) CRH levels at 28-30 weeks' gestation significantly and negatively predicted gestational length after ad- justing for antepartum risk. Moreover, subjects who delivered preterm had significantly higher CRH lev- els early in the third trimester than those who deliv- ered at term. In deliveries preceded by spontaneous onset of labor, maternal third-trimester CRH levels significantly and independently predicted earlier on- set of labor and preterm labor, whereas in deliveries effected by induction of labor or cesarean section, maternal CRH levels were a marker of antepartum risk but not an independent predictor of gestational length. Thus these findings support the premise that placental CRH may be implicated in the timing of human delivery in at least two ways. Firstly, pla- cental CRH may play a role in the physiology of parturition. Therefore premature or accelerated acti-

vation of the placental CRH system, as reflected by precocious elevation of maternal CRH levels, may be associated with earlier onset of spontaneous labor and resultant delivery. Secondly, placental CRH may be a marker of antepartum risk for preterm delivery, and therefore an indirect predictor of earlier delivery (Wadhwa et al., 1998a).

Biobehavioral studies of the human fetus

The developing human CNS may be more vulner- able to environmental perturbations than any other system because it develops over a much longer pe- riod of time (11-12 years); it has limited repair capabilities; its units have highly specific functional roles; the blood-brain barrier is not fully developed in utero; and the programming of neurotransmitter systems during critical developmental periods affects the organism's response to all subsequent experience (Rodier et al., 1994). However, the influence of the maternal and intrauterine environment on the devel- oping human fetal brain is poorly understood. This is partly because the assessment and quantification of human fetal brain development represents many theoretical and methodological challenges (DiPietro et al., 1996a). We have performed three studies in order to quantify and examine the influence of the fetal environment on brain development.

The first study was performed on a sample of 84 fetuses at 31-32 weeks' gestation to examine the ability of the fetus to learn and recall infor- mation. Three series of vibroacoustic stimuli were presented at pseudo-random intervals over the fetal head, and FHR responses to the first series of 15 stimuli (S1) were compared with responses to an identical second series of 15 stimuli (S1) separated from the first set by the administration of a single novel stimulus of different intensity and frequency ($2). A significant habituation pattern of responses was observed across trials for both series of stimuli, but this habituation pattern was attenuated for the series following the novel stimulus. These findings suggest that the 32-week-old human fetus may be capable of detecting, habituating and dishabituating to an external stimulus, and support the premise that areas of the human fetal CNS that are criti- cal for some aspects of learning and memory have developed by early in the third trimester (Sandman

et al., 1997). In a sub-sample of 33 mother-fetus pairs from the above study, the relationship between maternal (placental) levels of CRH and this pattern of fetal habituation and dishabituation in response to external stimulation was examined. The results indicated that the fetuses of mothers with highly el- evated CRH levels did not respond significantly to the presence of the novel stimulus, thereby providing preliminary support for the notion that abnormally elevated levels of placental CRH may play a role in impaired neurodevelopment, as assessed by the degree of dishabituation (Sandman et al., 1999).

We have recently performed non-linear statistical analyses on our complete sample of 156 mother- fetus pairs studied at 31-33 weeks' gestation. These analyses of FHR arousal and reactivity data, using a non-linear repeated-measures model with auto- correlated errors within subjects and independence across subjects, showed the following. Fetuses ex- hibited a significant, non-linear increase in FHR in response to the vibroacoustic stimulation protocol. After an initial response period, fetuses exhibited a FHR response decrement to subsequent stimuli, indicating habituation. Baseline FHR, presence of uterine contractions during trials, and characteris- tics of the challenge protocol such as inter-trial interval significantly influenced the magnitude of FHR responses. After accounting for these vari- ables, maternal conditions related to psychological and physiological stress (i.e. psychosocial stress lev- els, placental CRH concentrations, umbilical blood flow, and presence of maternal medical risk con- ditions) were significantly associated with the pat- tern of FHR responses. Habituation rate, assessed in a two-parameter growth curve (power) model, ac- counted for approximately 70% of the variance in FHR response. Fetal sex and conditions related to maternal stress (i.e. maternal ACTH concentrations, presence of medical risk conditions) were signifi- cantly associated with the rate of habituation; thus these matemal factors are predictive for the differ- ences between individuals in the patterns of fetal responses to external challenges (Wadhwa et al., 1999). This set of findings provides further support for the role of prenatal environment in modulat- ing some aspects of human fetal brain development related to recognition, appraisal, response, memory and habituation.

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Prenatal factors and infant developmental outcomes

To date, a very small number of human studies have been conducted to examine the effects of prenatal stress on subsequent infant development. Moreover, many of these studies suffer from methodological limitations such as retrospective design and inade- quate control of covariates. Therefore we conducted preliminary studies to assess the relationship of ma- ternal stress and stress hormones during pregnancy and delivery with indices of infant neuro-develop- ment. The study samples consisted of 47 mother- infant pairs 6 weeks after delivery, 24 mother-infant pairs 4 months after delivery, and 49 mother-infant pairs 3 years after delivery. All mothers had been participants in our above-described studies of pre- natal stress, fetal development and birth outcomes. Questionnaires were administered to mothers of 6- week-old infants to assess infant temperament and to mothers of the 3-year-old infants to assess behavioral characteristics of toddlers, including activity level, anger proneness, and social fearfulness. In collabo- ration with Kagan and colleagues, a laboratory-based behavioral assessment protocol (the Harvard Infant Behavioral Reactivity Protocol, Kagan and Snidman, 1991) was administered to the 4-month-old infants to measure infant motor and cry reactivity to a series of visual and auditory challenges. After adjusting for the effects of maternal influence, intra-partum compromise and prematurity, the overall pattern of results yielded support for the study hypotheses that higher levels of prenatal stress and stress hormones were significantly associated with an infant's tem- peramental difficulties at age 6 weeks, 4 months, and 3 years. Moreover, in utero measures of fetal arousal and reactivity also significantly predicted infant tem- peramental difficulties in these infants (Snidman et al., 1998; Wadhwa et al., 1998b).

Summary and conclusions

Our findings to date support a significant and inde- pendent role for behavioral processes such as prena- tal stress in the etiology of prematurity-related out- comes, and suggest that these effects are mediated, in part, by the maternal-placental-fetal neuroendocrine axis. Our findings also suggest that the use of a fetal challenge paradigm offers a novel way to quantify

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fetal neurologic maturity in utero, and that the ma- ternal environment exerts a significant influence on the fetal autonomic nervous system as well as central brain processes related to recognition, memory and habituation. Finally, our findings provide preliminary evidence to support the notion that the influence of prenatal stress and maternal-placental hormones on the developing fetus may persist after birth, as as- sessed by measures of temperament and behavioral reactivity in the first 3 years of postnatal life.

Our findings are consistent with our hypothesis and also with the work of several other research groups. In the past few years, several large, popula- tion-based epidemiological studies of prenatal stress and prematurity-related outcomes have suggested that high levels of maternal psychosocial stress are associated with a 1.5- to 2.5-fold increase in the adjusted risk for prematurity (Goldenberg et al., 1991; Cliver et al., 1992; Hedegaard et al., 1993, 1996; Pritchard and Teo, 1994; Copper et al., 1996; Nordentoft et al., 1996). Studies of physiological reactivity in pregnancy have reported attenuated re- sponses to exogenous challenges. For instance, preg- nancy has been associated with blunted autonomic and endocrine responses to a variety of physical and chemical challenges (Nisell et al., 1985a,b; Barron et al., 1986; Goland et al., 1990; Schulte et al., 1990; Matthews and Rodin, 1992). Longitudinal investiga- tions of the functional development of the human fetal central nervous system over the course of gesta- tion have indicated that chronic maternal psycholog- ical distress is significantly related to indices of fetal neurobehavioral maturation. Specifically, greater ma- ternal stress was found to be associated with reduced fetal heart rate variability and reduced coupling be- tween fetal heart rate and movement (DiPietro et al., 1996a,b). Other research groups have demonstrated that placental CRH is involved in the physiology of normal parturition and that elevated CRH con- centrations significantly predict risk for spontaneous preterm birth (Campbell et al., 1987; Wolfe et al., 1988, Warren et al., 1992; McLean et al., 1995; Ko- rebrits et al., 1998b; Hobel et al., 1999). Lastly, the small number of studies that have examined the ef- fects of prenatal factors on infant development have reported significant associations of maternal prenatal stress with newborn and infant temperament (Zuck- erman et al., 1990; Martin et al., 1997), with in-

fant neurodevelopmental disorders (Mclntosh et al., 1995; Ward, 1990) and with infant psychopathology (Ward, 1991).

Although this growing body of work by our group and others indicates that the prenatal environment may influence the development of the fetus and subsequently related outcomes, several questions re- main. These questions relate to outcome-specificity, exposure-specificity, the timing of exposure during gestation, and the biological and behavioral mech- anism(s) by which the prenatal environment may act upon the developing fetus. We are particularly interested in the importance of the timing of stress exposure during gestation, so we are serially assess- ing matemal and fetal responses to behavioral stress at three time points in early, mid and late gestation to determine whether there are specific periods of increased or heightened fetal vulnerability. We aim also to understand the biological mechanisms un- derlying the effects of stress on neural development. Maternal infection has been identified in the obstetric literature as being a particularly important risk factor in the etiology of extreme prematurity, and perhaps also in certain aspects of sub-optimal fetal brain de- velopment (Romero et al., 1994). We are exploring the hypothesis that maternal stress may negatively influence immune mechanisms, by altering the bal- ance between Thl and Th2 lymphocyte responses, thus increasing maternal and fetal vulnerability to pathogen exposure.

The importance of the types of research out- lined above cannot be over estimated. Prematurity is the leading cause of infant mortality and morbid- ity in the non-anomalous fetus in the United States. The prevalence of prematurity is higher in the USA than in any other developed nation in the world and has not decreased significantly over the last 40 years. The etiology of prematurity is unknown in one-half to two-thirds of all cases and preven- tion programs to reduce the incidence have largely been unsuccessful (Creasy, 1994). Although the vast majority of premature newborns survive, studies of short-term outcomes find significantly higher rates of severe morbidity in the neonatal period, including asphyxia, meconium aspiration, hypoglycemia, poly- cythemia and respiratory distress syndrome. Fur- thermore, studies of long-term outcome have found higher rates of sensorineural impairments and dis-

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abilities (e.g. cerebral palsy, and visual, auditory and intellectual impairments) and higher rates of com- plications affecting the respiratory, gastrointestinal and renal systems (Blair and Stanley, 1992; Low et al., t992; Knoches and Doyle, 1993). In the USA over 48 million people, or 15% of the population, suffer from some form of neurological disorder, not including mental disease. A large proportion of these disorders are believed to originate during prenatal life, and elucidating their etiology presents a per- plexing neurological problem (Kolb, 1995).

Another implication of this research relates to the risk of developing chronic degenerative disease in later life. Recent research suggests that 'set-points' for homeostatic feedback and control mechanisms are determined during intrauterine life and can there- after alter the individual's response characteristics to her or his environment throughout life. From this work a model has been proposed by which human disease is programmed during fetal life. (Barker, 1998). Given that a fetus is most vulnerable dur- ing periods of rapid cell division, it has also been argued that humans are more vulnerable to the nox- ious effects of an adverse intrauterine environment due to the rapidity and complexity of human growth and development in utero. Epidemiological evidence from several recent population-based studies in Eu- rope, North America, and Asia lends support for this hypothesis. It appears that pre- and perinatal pro- cesses, as indicated by measures of growth and size at birth and during early life, significantly predict the risk of chronic degenerative diseases such as hy- pertension, coronary artery disease, and adult-onset diabetes mellitus over and above those conferred by established physiological and behavioral risk factors, i.e. the magnitude of the effect equals or exceeds that of all other risk factors combined together (Barker, 1998; Nathanielsz, 1999).

Some 60 years ago, the Fels study of early devel- opment, probably the first systematic investigation of factors that affect development before birth, sug- gested that "such factors as [maternal] emotional life and activity level during gestation may contribute to the shaping of physical status, behavioral patterns, and postnatal progress of the children they bear" (Sontag, 1941). Although we have come a long way since then, the continued investigation of the inter- face between biology and behavior in prenatal life

promises to realize the full implications of this state- ment with respect to the nature of our origins and for our health and well-being.

Abbreviations

ACTH CNS CRH FHR GABA HPA mRNA SAM

adrenocorticotropin central nervous system corticotropin-releasing hormone fetal heart rate gamma aminobutyric acid hypothalamo-pituitary-adrenal messenger ribonucleic acid sympathetic-adrenal-medullary

Acknowledgements

The authors would like to gratefully acknowledge the invaluable contributions of our collaborators and colleagues to this program of research: Aleksan- dra Chicz-DeMet, Ph.D., Christine Dunkel-Schet- ter, Ph.D., Laura Glynn, Ph.D., Kimberly Herbel, M.A., Calvin J. Hobel, M.D., and Manuel Porto, M.D. These studies were supported, in parts, by US PHS (NIH) Grants HD-33506, HD-28413, and HD-28202.

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