Third, variations in the fetal physiological environment, caused by the aforementioned external factors, appear to have effects on brain development, which may lead to neonatal brain disease or psychopathology later in life. In the field of child neurology, etiological factors for cerebral palsy are sought in the prenatal period, because perinatal complications seemed to explain only part of the occurrence of this brain dysfunction in at term born infants (Bottos et al., 1999; Casaer, 1993; Sugimoto et al. , 1995; Truwit et al., 1992).The harmful effects of prenatal exposure to tobacco, alcohol, medication or drugs on human brain development are well established (see for a review: Ferreiro & Dempsey, 1999). In short, gestational alcohol exposure may result in microcephaly and central nervous system 11 From postnatal to prenatal determinants of development malformations (Sokol & Clarren, 1989; Day & Richardson, 1991), disturb neuronal migration (Day & Richardson, 1991) and reduce neuronal numbers (Kumari & Ticku, 1998; Pantazis et al., 1998). According to these studies, alcohol exerts its effects on a variety of genes and can modify the composition of the postsynaptic membrane (Ferreiro & Dempsey, 1999). Likewise, fetal nicotine exposure results in alterations of cholinergic, noradrenergic and dopaminergic projections in postnatal life and appears to elicit a premature switch from proliferation to differentiation. In addition, fetal cell damage and cell loss is found (Slotkin, 1998). Besides the direct effect of alcohol, nicotine and cocaine on fetal brain development, the prenatal exposure to these substances may further interfere with normal brain processes. This may result in later cognitive dysfunction (Fried et al., 1998; Richardson et al., 1995; Frydman, 1996; Naeye & Peters, 1984), behavioral deficits (Weissman et al., 1999; Eckhardt et al., 1998; Fergusson et al., 1998; Williams et al., 1998; Olson et al., 1997; Sampson et al., 1997; Wakschlag, et al. , 1997; Orlebeke et al., 1997; Milberger et al., 1996) and even mental retardation (Drews et al., 1996). Exposure to high levels of radiation in pregnancy has also been related to mental retardation (Otake & Schull, 1984). Other prenatal factors linked with later compromised development, in particular schizophrenia and major affective disorders, included malnutrition (Hoek et al., 1998; Susser et al., 1998; Geddes, 1999) and influenza (Machon et al., 1997). The effects of these external prenatal factors generally result in explicit physical pathology, growth retardation or psychopathology. Internal prenatal factors such as maternal stress may lead to a more subtle disruption in normal development besides the obvious effects on general fetal development that resulted in preterm birth and lower birth weight. In humans, prenatal stress has been associated with a smaller head circumference of the neonates (Lou et al., 1994). Severe maternal stress in pregnancy has been linked with an increased risk for schizophrenia at adult age for the infants born of these mothers (Van Os & Selten, 1998). In nonhuman primates, it was found that prenatal stress had long-term effects on behavioral regulation in the offspring that persist into adolescence (Clarke & Schneider, 1997). Offspring of prenatally stressed rats had stronger and prolonged responses of the Hypothalamic-Pituitary- Adrenal (HPA) axis in stressful situations (Weinstock, 1997). The effects of prenatal stress on animal offspring will be discussed in detail elsewhere in this thesis.

     The early effects of stress hormones on the developing brain may account for a part of variation in human brain development. So far, the effects of psychosocial factors, and the direct or mediating effects of stress hormones and gonadal hormones on fetal brain development have received little attention from the field of infant studies until now. Therefore, this chapter aims to affirm that it is worthwhile to study the influence of prenatal determinants, and in particular of prenatal stress, on infant development. The concepts of early programming and prenatal maternal stress are briefly introduced. Fetal brain development and possible sensitive periods in fetal brain developement are described. The role of prenatal hormones as mediators for the effect on human development is elaborated. Moreover, the importance of longitudinal, prospectively designed studies is endorsed. 

1 . 2 . 2 T h e c o n c e p t o f p r e n a t a l m a t e r n a l s t r e s s

include some forms of schizophrenia (Korelman & Scheibel, 1983) and of dyslexia (Sherman et al., 1985). Middle gestation is known as a period during which there is neuroblast proliferation and adult numbers of neurons are virtually achieved by the time it is over (Dobbings & Sands, 1979). The later part of gestation corresponds to the brain growth spurt, a period during which brain weight and developmental processes proceed very quickly, which continues 13 From postnatal to prenatal determinants of development postnatally, and to early synaptogenesis. The timing of brain development differs from one cell type to another and from region to region, however, resulting in an extremely complex developmental pattern.

Even small variations in the fetal hormonal environment have been shown to exert programming effects on the developing brain. Especially the internal steroid hormones produced by the gonads, the thyroid, and the adrenal glands have been found to exert a great influence on the development of the fetal brain (Collaer & Hines, 1995; Sikich & Todd, 1988). Steroid hormone effects occur at the level of gene transcription, via the actions of intracellular hormone receptors. Other effects occur at the level of the membrane via receptors on the cell surface that produce rapid effects on bioelectrical activity and secondary messenger systems. These hormones are regarded as links between the gene and the environment (McEwen, 1992). Increased or decreased levels of maternal steroid hormones during pregnancy may be the mediating factors in explaining disruptions in development. First, products of the gonads, such as androgens, estrogens and progesterone have shown to program the development of the brain and to affect social development of the infant. Higher levels of testosterone were associated with timidity in preschool boys (Marcus et al., 14 Chapter 1 1985) and increased visual-spatial performance in girls at the age of 6 years (Jacklin & Maccoby, 1988). More recent studies (Finegan et al., 1992; Grimshaw et al., 1995) found indications of an effect of gonadal hormones on the cerebral lateralization and of cognitive abilities at 4-year-old girls. Prenatal testosterone levels showed a curvilinear (inverted U-shape) relation to language comprehension and classification abilities, and a linear relation to counting, number facts and block building. Furthermore, girls with higher prenatal testosterone levels were more strongly right-handed and had stronger left-hemisphere speech representation, thus had a greater lateralization of function than girls with lower prenatal testosterone levels. Second, thyroid hormone levels were found to be critical signals for brain development. The presence of sufficient levels of maternal thyroid hormones during the first 10-12 weeks of gestation is important because only from that period on does the fetus start to produce its own thyroid hormones. It has been shown that subtle and subclinical abnormalities of maternal thyroid status in this early period in pregnancy were associated with a compromised cognitive development at age five years (Pop et al., 1995). Finally, the adrenal gland produces hormones, like cortisol, that are involved in stress responses. From animal studies we know that prenatally stressed rodents and monkeys showed an abnormal reactivity of the Hypothalamic-Pituitary-Adrenal (HPA) axis early and later in life. Also, prenatal exposure to prolonged increased levels of corticosteroids resulted in adverse social and exploratory behavior and motor developmental delays in offspring. Studies of Mary Schneider et al. (1992a,b,c; 1993; 1997; 1999) showed that prenatal stress (daily exposure to unpredictable noise stimuli) in nonhuman primates was associated with slower motor development, less exploration in a novel environment and more disturbed behavior of the offspring, even in the absence of clear effects on physical  development. In short, small variations in maternal hormone levels during gestation may result in developmental problems later in life. Therefore, these variations in maternal hormone levels may be mediating the effect on brain development. In this thesis the focus will be on the potential mediating effects of maternal stress hormones, in particular the HPA axis activity, on postnatal developmental delays and behavioral problems.

1 . 2 . 6 M e t h o d o l o g i c a l g u i d e l i n e s

Precise analysis of the relationship between prenatal events or elevated stress hormones and disruption of fetal brain development and later deviant development is very difficult, especially in humans. Many factors may confound assignments of causation to specific agents. Increased prenatal stress hormones is often accompanied by exposure to prenatal alcohol or nicotine. Furthermore, intrinsic genetic variation could influence the susceptibility of the mother or fetus to potential harmful hormones, which may further complicate efforts to identify factors that are harmful for the developing brain. In this field of research, retrospectively designed studies are commonly used in order to examine possible prenatal stress influences on later compromised development (Van Os & Selten, 1998; Huttunen et al., 1994; Clements, 1992; Meier, 1985; Stott, 1973; Davids, 1963). Since most of these retrospective studies depend on maternal reports, the effects cannot be attributed to specific forms and amounts of stress and confounding factors cannot be adequately controlled for. Recall bias is 15 From postnatal to prenatal determinants of development a major disadvantage of such a design. Moreover, it is almost impossible to study the effects of a particular factor in a specific period during pregnancy. Prospective longitudinal studies are therefore needed to collect data throughout pregnancy at several gestational periods and from birth onwards. In the present thesis we therefore used a prospective longitudinal design. It is important to appreciate that the design of the present studies does not allow to examine the relative contribution of genetic versus environmental versus gene by environmental interactional influences on infant development and temperament. Twin studies in humans have shown that the exposure to stressful life events and daily hassles is not independent of the genetic make-up of the individual (Kendler et al., 1993). Especially, for personal and network dependent stressful life events a significant aetiological role for genetic factors has been found (Kendler et al., 1999). Therefore, any association between stress of the pregnant mother and infant development cannot simply be interpreted as reflecting environmental influences on infant development. Rather, these assocations will be due to a complex interplay between genetic and environmental influences that can be unraveled only by using genetic sensitive designs like twin and (half-)sibling studies.

1.3 Conclusion

The concept of early programming of the development of behavior and cognition with a focus on prenatal instead of postnatal influences offers a new paradigm for researchers interested in infant and child development. Even small variations in fetal physiological environment in specific periods of time may be mediating the effects on later development. It is still questionable if specific sensitive periods exist for the harmful effects of prenatal exposure to maternal stress hormones. Prospective longitudinal studies will offer the opportunity to investigate the effects on the developing fetal brain more precisely. Researchers in the field of infant studies, should take advantage of the knowledge already available in other fields of research and start studying children from their earliest days, instead of having birth as a starting point of their focus of attention. Since prenatal stress is common in today's and probably tomorrow's society, we strongly recommend research with a focus on the effects of psychosocial factors, such as stress, on development from the earliest stages of human life onwards. 16 Chapter 1

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