Sex Steroids at Birth: Genetic and Environmental Variation and Covariation LAURA M. SAKAI LAURA A. BAKER CAROL NAGY JACKLIN University of Southern California Los Angeles, California
IRA SHULMAN Los Angeles CountylUSC Medical Center Los Angeles, California
Three sex-steroids (estradiol, progesterone, & testosterone) were assayed from the umbilical cord blood of 58 same-sex twin pairs in an investigation of the effects of sex, as well as genetic and environmental factors, on neonatal hormone levels. Although significant mean differences were found between boys and girls for both testosterone and progesterone, sex appeared to account for very little of the total variation for any of the hormones. Results showed that genetic influences significantly affected within-sex variation in both estradiol and progesterone levels, while variations in the intrauterine (shared twin) environment accounted primarily for differences in levels of testosterone. Moderate correlations were also found among the three hormones. Multivariate biometrical analyses revealed these relationships to be explained by an underlying general factor of nonshared environmental influences affecting all three hormones. Genetic factors appeared to be specific to each hormone rather than correlated across hormones. These results suggest not only that genes are operating at this early age, but also that maternal and other prenatal factors (e.g., placental effects, uterine position) have a significant role in variations of sex-steroids and possibly on later behaviors.
Psychologists have recently become very interested in the individual differences in sex-steroid hormones and their role in anatomical, functional, and behavioral development. Although some sex differences in certain hormones do exist, which may in turn relate to some sexually dimorphic characteristics, there is also considerable variation within males and females for the same sex-steroid hormones (e.g., Marcus, Maccoby, Jacklin, & Doering, 1985; Jacklin, Maccoby, Doering, & Reprint requests should be sent to Laura A. Baker, Department of Psychology, SGM 501, Los Angeles, CA 90089-1061, U.S.A. Received for publication 1 1 December 1989 Revised for publication 14 August 1991 Accepted at Wiley 9 September 1991 Developmental Psychobiology 24(8):559-570 (1992) 0 1992 by John Wiley & Sons, Inc.
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King, 1984; Maccoby, Doering, Jacklin, & Kraemer, 1979). To date, however, the causes of these individual differences and the mechanisms underlying their relationships to psychological and physical characteristics remain unknown. Both the nervous system and the endocrine system are recognized as control mechanisms that regulate hormone behavior relationships. This control begins early during fetal development. The human brain grows most rapidly between the middle of gestation through the second year after birth. During this development, there are critical periods during which the brain is most susceptible to the presence of particular hormones. Gonadal hormones can imprint a functional pattern on the brain during this critical period which influences future patterns of behavior. For example, brain development begins as fundamentally female. However, the presence of androgen produced by the testis will impose a masculine pattern of development on the fetus. Sex hormones can thus play an organizing role on the physical development of the fetus and are quite influential even prenatally. The organizing effects of hormones on human development has been generalized to include hormonal influences on individual differences in cognitive abilities and sexually dimorphic behaviors (Wilson & Foster, 1985). Sex steroids are important determinants of certain differences between males and females, such as genitalia differentiation (Wilson, George, & Griffin, 198 l), brain organization, and sexual behavior (see Hines, 1982). In addition, the nature of sex differences may change at different times in the life cycle. That is, sexsteroid levels of men and women differ markedly during puberty, but less overlap may be seen in early fetal life. In recent years, however, it has become increasingly evident that there are substantial variations of these same hormones within each sex. It is no longer viewed that there are exclusive male or female hormones, per se, but that both sexes possess the same hormones, albeit in varying degrees. Sexual differentiation is rather the result of differences in levels of individual hormones in males and females, rather than the presence or absence of certain hormones (Wilson & Foster, 1985). These within-sex hormonal variations have also been associated with individual differences in cognitive performance and social behavioral development. Most surprisingly, neonatal sex hormones appear to be the chemical mediators of some sexually dimorphic human behaviors displayed at later ages. For example, individual differences in testosterone and androstenedione in umbilical cord blood have been reported to be significant predictors of spatial ability in 6-year-old girls, while timidity scores in 6- to 18-month-old boys were related to neonatal levels of progesterone, testosterone, and estradiol (Jacklin, Maccoby, & Doering, 1983; Jacklin, Thompson-Wilcox, & Maccoby, 1988). These provocative findings suggest that patterns of stereotypical masculine or feminine behaviors may be partly biologically based rather than solely a result of social learning. However, it must be emphasized that within-sex variations appear far greater than between-sex differences in sex hormones (Maccoby, et al., 1979; Hines, 1982) as well as in most sexually dimorphic behaviors (Maccoby & Jacklin, 1974). Thus, an important step in our understanding of hormone-behavior relationships must be to realize the sources of these individual differences in sex hormones in males and females. While previous research has implicated the role of circulating maternal hormones and parity factors in neonatal sex-steroid levels (e.g., Maynard, Heyes, & Shaxted, 1982; Shutt, Smith, & Shearman, 1974), these only account
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for a small portion of variation of hormone levels at birth. The importance of other characteristics of the uterine environment, as well as of the fetus itself, remain important questions in our understanding of both within- and between-sex differences in sex-hormone levels. Genetic variations among individuals are included in the most important classes of phenotypic or observed variance in most behavioral and physical characteristics. However, the role of genetic factors in individual differences in sexsteroid levels has yet to be studied. The twin design provides a basis for which questions concerning fetal genetic, uterine environment, and other maternal factors may be addressed. Hormonal assays were performed on umbilical cord blood at birth for 36 monozygotic (MZ) and 22 dizygotic (DZ) twin pairs. Although these cord-blood samples are reflective of maternal, fetal, and placental units, these various effects may be considered in a genetic and environmental framework of individual differences. For example, maternal effects should produce similarities between co-twins, regardless of their zygosity (MZ or DZ), thus reflecting shared environmental influences. On the other hand, to the extent that placental units produce differences between co-twins, these would be represented as unique or nonshared environmental effects. Finally, genetic variations in fetal units will produce less observed similarities in DZ twin pairs, who share only half their nonsegregating genes, as compared to the genetically identical MZ twins. In addition to parsing hormonal variation into genetic, shared environmental, and nonshared environmental sources, the twin design allows genetic and environmental covariation among several hormones to be investigated through multivariate biometrical analyses. Such analyses are necessary in the present study since neonatal levels of these sex steroids have been previously shown to be positively correlated, indicating a lack of independence among them (Marcus et al., 1985). For example, it is recognized that testosterone serves as a substrate for estradiol. The observed correlation between these steroid levels at birth may be due to correlated genetic influences, correlated environmental influences, or both. A genetic correlation may relfect linkage disequilibrium and/or pleiotropic (multiple) effects of the same genes on the two hormones. Similarly, environmental correlations among the hormones would point to some aspect(s) of the maternal environment which influence levels of two or more sex steroids.
Method Design This study used a multivariate twin design to investigate inherited (genetic) and noninherited (environmental) influences on levels of estradiol, progesterone, and testosterone in umbilical cord blood samples taken at birth. Radioimmunoassays were performed on plasma samples of both monozygotic (MZ) and dizygotic (DZ) twins. The similarity between twins for each of the three hormones was then assessed. The twin design is based on the knowledge that MZ twins share 100% of their genes while DZ twins share, on the average, 50% of their segregating genes. If genetic factors influence a given trait, such as the level of estradiol, MZ twin pairs are expected to demonstrate significantly greater phenotypic similarity as
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compared to DZ pairs. Intraclass correlations may be calculated separately for MZ and DZ twins using within- and between-pair mean squares (MSW, MSB) as follows:
'
MSB - MSW = MSB + MSW
Heritability (h2),or the proportion of observed (phenotypic) variance explained by genetic factors, can be roughly estimated by doubling the difference between the within-pair correlation for.MZ twins and that for DZ twins (Falconer, 1981). That is,
h2 = 2(rMz - rDz). Proportions of variance explained by environmental factors common to members of the same twin pair ( c 2 )and by specific environmental factors unique to each individual (s *), can also be obtained algebraically from these zero-order, isomorphic correlations (rMzand rDZ). In multivariate analyses presented in this article, of additional interest are the cross-correlations between a given trait in one twin, such as his or her estradiol level, with a different trait in the co-twin, such as the level of testosterone. If genetic factors for estradiol are correlated with genetic factors for testosterone, this cross correlation is expected to be of greater magnitude in MZ twin pairs than in DZ pairs. However, if correlated environmental influences are the primary source of covariation between estradiol and testosterone levels, then cross-correlations should be comparable for MZ and DZ pairs. The extent to which correlated genetic and correlated environmental influences contribute to the phenotypic correlation between two traits, X and Y , may be readily seen in the following equation:
where hx and h, are square roots of heritabilities for traits X and Y respectively, cx and cy are square roots of common environmentalities for the two traits, and sx and sy are square roots of specific environmentalities. The cross-correlation bewhile rCXyand tween genetic influences for the two traits is symbolized by rGxyr rsxyrepresent the cross-correlations between environmental influences common to the two twins and between environmental influences specific to each twin. The LISREL-VI program (Joreskog & Sorbom, 1983) was used to estimate all of these genetic and environmental parameters simultaneously, as described by Fulker, Baker, and Bock (1984) and Ho, Baker, and Decker (1988). The procedure requires input matrices of between- and within-pair mean squares and cross products among the observed variables and yields constrained maximum-likelihood estimates of heritabilities, environmentalities, genetic correlations, and environmental correlations as described above. That is, observed variances and covariances are decomposed into diagonal matrices of square roots of heritabilities [h], common environmentalities [cl, and specific environmentalities [s] . Additional
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563
matrices contain genetic correlations [RG], common environmental correlations I&], and specific environmental correlations [R,]. Results from biometrical analyses will be presented in this more efficient matrix notation. Various models testing the relative influence of genetic and environmental sources of variance and covariance will be presented along with goodness-of-fit statistics.
Subjects The subjects consisted of 58 same-sex twin pairs (i.e., 116 infants) born at the Women’s Hospital on the USC Health Sciences campus. This hospital primarily serves women of low income as well as families on welfare. Twins included in this study primarily were of Hispanic descent and in good health. For the majority of twin pairs (96%), the gestational age exceeded 34.5 weeks and the birthweight of each co-twin exceeded 2250 g. These criteria are well within the normal range in the population of twin births (Vlietinck et al., 1989). Therefore, although the population might be considered at risk because of their poverty status, we sampled twins with an average birthweight that is considered high for twins in attempt to screen out other possible health complications. Birthweight was not found to be significantly related to hormone levels. Gestational age was significantly related only to levels of testosterone with older babies having more testosterone detected in their umbilical cord blood (r = .32, p < .05). Zygosity (MZ or DZ) was determined through antigen typing at the blood bank at the Los Angeles County/USC Hospital. For each twin, the presence or absence of 18 different antigens (A, B, D, C, E, C, G , M, N, K, E, Kpb, Fy”, Fyb, Jk”, Jkb, s, s), was determined. If a twin pair did not show an exact antigen match, they were considered DZ. On the other hand, twin pairs that showed an exact match of all 18 antigens were considered MZ. Although there is a remote chance that some of the pairs classified as MZ could conceivably be DZ, such misclassifications would not seriously affect our results. This procedure resulted in classification of 36 MZ and 22 DZ twin pairs.
Measures and Procedures Recent mothers of twins were reached through the Los Angeles County Women’s Hospital and informed consent was obtained to perform the red blood cell antigen-typings and sex-steroid radioimmunoassays on their twins. At the time of birth, 7-10 ml of umbilical cord blood was obtained for each twin and allowed to clot in a refrigerator. The serum was separated and then frozen at -20°C. Radioimmunoassays were subsequently performed on the frozen serum samples for three sex steroids: estradiol, progesterone, and testosterone. Progesterone kits were used to Extraction lZsIEstradiol and extraction 1251 measure levels of estradiol and progesterone respectively. These kits were purchased from Pantex in Santa Monica, California. The lowest detectable weight for estradiol was 10 pg/ml in serum. Intraassay coefficient of variation was 8% and interassay coefficient of variation was 10.5%. Sensitivity for progesterone was .5 ng/ml. Intraassay coefficient of variation was 7.5% and the interassay coefficient of variation was 10%. Levels of testosterone were determined with a Testosterone Radioimmunoassay (RIA) kit from the Diagnostic Products Corporation in Los
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Table 1 Means (SD) c$ Hormone Levels (nglml) by Gender and Zygosity Male Infants
Estradiol Progesterone* Testasterone*
Female Infants
MZ
DZ
( n = 46)
(n = 20)
9.15 (4.54) 621.23 (267.52) .69 ( .24)
10.33 (3.78) 678.90 (320.22) .82 (.19)
MZ (n = 26)
( n = 24)
DZ
8.15 (4.63) 465.73 (291.22) .55 (.I91
10.11 (6.91) 585.54 (264.98) .58 (.20)
“Indicates significant difference between males and females ( p 5 .05).
Angeles, California. Sensitivity for testosterone was 10 pg/dl and the intraassay coefficient of variation was 9.7%. Specificities for the various tests can be obtained from Pantex and Diagnostic Products Corporations. Assays were performed at the LA County/USC Endocrinology Lab twice for each sample to assess test-retest reliability.
Results Descriptive Statistics Hormone levels were calculated twice for each twin and test-retest reliability was then assessed for each hormone. Reliability coefficients were extremely high for all three hormones: testosterone ( r = .95), progesterone (r = .96), estradiol ( r = .99). Thus, individual hormone levels were calculated as the average level based on two separate assays. Mean hormone levels are reported by gender and zygosity in Table I . Means levels were consistently higher for DZ twins than for MZ twins although these differences were not statistically significant. Progesterone and testosterone levels were significantly higher for boy neonates than girl neonates ( p < .05). There were no significant effects of Zygosity nor any Sex x Zygosity interactions. Since there were significant differences between boys and girls for levels of both progesterone and testosterone, these sex effects were statistically removed from raw scores of each hormone using a multiple regression procedure to eliminate any spurious associations among the steroids due to their shared sex variation. Residual scores from the multiple regression of each steroid on sex were next computed. These residual scores were then transformed into standardized z scores with zero means and unit variances. All subsequent analyses presented here are based on sex-adjusted scores. As expected, there were moderate but significant correlations among the three sex-steroid levels (see Table 2), with correlations similar in magnitude and direction to those reported by Marcus and colleagues (1985). These interhormone relationships, or lack thereof, may be understood in part by what is currently known about the chemical processes involved in hormone production. That is, since testosterone
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Table 2 Correlations among Sex Steroids Estradiol Estradiol Progesterone Testosterone
1 .o .31* .41*
Progesterone
Testosterone
1 .o .11m
1 .o
*p < .0001 < .lo.
"p
serves as a substrate for estradiol, a correlation between levels of these hormones is expected whereas no correlation might be expected between testosterone and progesterone since they do not serve as a substrate for one another in humans. However, what is of interest is not simply whether these relationships exist but rather the underlying etiologies of these relationships. For example, are levels of testosterone correlated with levels of estradiol because the same sets of genes are influencing both steroids or are different genes at work?
Biometrical Analyses In order to investigate inherited and noninherited influences in the variation and covariation of each of these three hormone levels, multivariate biometrical analyses were performed. Several models were fit to 3 x 3 between- and withinpair mean squares and cross-product matrices among levels of estradiol, progesterone, and testosterone, computed separately for MZ and DZ twin pairs. Goodnessof-fit statistics for the various models are summarized in Table 3. First, a full model containing matrices [ h l , [cl, [sl, [&I, [ R , ] , and [ R , ] was fit to the data (Model I). This allowed both inherited and noninherited influences as well as correlations among the hormones within each class of influences to be estimated. The full model yielded a nonsignificant chi square, ~ ~ ( 1 = 2 )8.82, p = .18, indicating that this model with correlated genetic and correlated environmental factors fit the data quite well. Models constraining one or more of the estimated parameters to be zero were next fit to the data. These constrained models allowed us to test the relative importance of inherited versus noninherited influences. First, [ h ] and [R,] matrices were constrained to be a zero matrix. That is, in this model, genetic influences were set to zero and any twin similarity must thus be accounted for by common environmental variance. Test of this model yielded a significant chi square, ~ ~ ( 1 2 ) Table 3 Biometrical Analyses of Estradiol, Progesterone, and Testosterone Model
x2
df
P
1. Full model 2. [hl = [RG] = 0 3. [c] = [R,] = 0 4. Final model
8.82 24.81 26.63 19.20
6 12 12
.18
15
.20
.02 .01
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Table 4 Maximum-Likelihood Parameter Estimates from Final Model Estradiol
Progesterone .72*
.68*
0
0
.32*
Testosterone 0
.46* .28*
.54*
1.oo
.62* *p
.31*
1 .OO
1
< .05.
= 24.81 ,p < .02, with a significant increase compared to the full model, x2difference (6) = 1 5 . 9 9 , < ~ .05, and an overall poor fit to the data. Therefore, genetic variances and/or covariances may account for a proportion of the observed twin similarity and must be included in the final model. Dropping common environmental influences ([c] and [R,] matrices; Model 3) also produced apoor fit to the data, ~ ~ ( 1 = 2 26.63, ) p < .02, indicating that the shared environment factors also play a significant role in the variation andlor covariation of sex-steroid levels. Although the full model yielded a nonsignificant chi square and a good fit to the data, several parameters in this model were found to be nonsignificant upon closer examination. These included genetic correlations among the hormones as well as the genetic variance for testosterone. In addition, variations in the common environment were negligible and nonsignificant for all hormones except testosterone. Covariation among intrauterine effects (i.e., common environmental correlations) for the three hormones were also nonsignificant. Thus, these nonsignificant parameters were constrained to be zero in the final model (Model 4). This final model yielded a nonsignificant chi square, ~ ' ( 1 5 ) = 19.20, p = .21, and a nonsignificant difference from the full model, x2 difference (9) = 10.38, p > .IO. Parameter estimates based on this final model are presented in Table 4. Since correlations among genetic factors and among shared environmental factors were all found to be nonsignificant in these data, observed relationships among these three hormones are entirely explained by correlations among nonshared environmental effects (i.e., R,). Furthermore, estimates from matrix [R,] appear to be uniform and suggested an underlying general factor affecting all three sex steroids. In addition, specific environmentalities (s2), heritabilities (h2),and common environmentalities ( c 2 )for each hormone were computed. As seen in Table 4, there were significant genetic influences for both estradiol and progesterone, although genetic influences appeared to be specific to each hormone rather than correlated across hormones. Genetic factors did not seem to influence testosterone levels. Variability in testosterone was instead mediated by intrauterine factors that are common to twin pairs ( c 2 = .46). The remaining phenotypic variance for each hormone was explained by environmental factors not shared by twins. This may include, for example, differences in the position of each twin in the uterus or placentation effects.
Discussion The present investigation of newborn twins therefore provides the first attempt to determine the relative contributions of sex, environmental, and genetic factors
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to variations in neonatal levels of three sex steroids: estradiol, testosterone, and progesterone. Hormonal assays were performed on umbilical cord blood at birth. Although these cord-blood samples are reflective of maternal, fetal, and placental units, their various effects may be considered in a genetic and environmental framework of individual differences. By comparing the observed (phenotypic) resemblance of MZ and DZ twin pairs for levels of hormones, effects of genetic factors operating in the fetus were separated from other maternal environmental influences. Furthermore, the extent to which the prenatal environment produced similarities or differences between co-twins were investigated. That is, a distinction was made between aspects of the environment which were shared or common to the twins (e.g., circulating nutrients in the womb, maternal hormone levels), and those which were not shared or are unique to each twin (e.g., position in the uterus). Differences in hormone levels between males and females were in the expected directions with boys having higher levels of testosterone and progesterone than girls. However, unlike other times during the life cycle (e.g., puberty), sex differences accounted for very little of the total variation of any of these neonatal hormones ( r 2 = .001, .037, and .129 for estradiol, progesterone, & testosterone, respectively). As is often found in studies of sex differences in behavior, there was substantially greater within-sex variation in sex hormones compared to betweensex differences. In addition, these three sex steroids were moderately intercorrelated. Environmental factors specific to each twin appeared to account for this covariation. Also note that these specific or nonshared environmental factors contributed significantly to individual differences in levels of each specific hormone. The significant role of these specific environmental factors in the variation and covariation of sex steroids was particularly interesting, especially since both MZ and DZ co-twins share the same uterine environments at the same time. In spite of this, there were apparently enough differences within a given pregnancy to account for substantial variation between the twins. These factors could be prenatal experiences unique to each twin such as position in the uterus and other placentation effects as discussed further below. Moreover, whatever the source of these within-uterus differences, they appeared to influence all three hormones simultaneously. Several environmental factors may be unique to each twin. One possibility comes from neuroendocrinology studies on nonhuman animals. Studies on rats give evidence that position in the uterus may influence the presence of androgens in littermates. Male rats located on the caudal side of the females appear to have a masculinizing effect, thus influencing the birth weight, mounting behavior, and responsiveness to the activational effects of testosterone (Babine & Smotherman, 1984; Meisel & Ward, 1981). The masculinizing effects of intrauterine, contiguous male littermates on female behavior have also been demonstrated with guinea pigs, mice, and hamsters (Gandelman, 1986; Kinsley et al., 1986; Tobet, Dunlap, & Gerall, 1982). Since this effect has been observed only in nonhuman animal research, it is difficult to directly compare this information to our study on same-sex human twins, and extreme caution must be taken when generalizing these effects. However, it does suggest that possible differences occur in what is typically viewed as a rather homogeneous uterine environment. That each individual twin has unique experiences within the womb appears highly tenable. Other evidence for prenatal differences between members of a twin pair come
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from studies on pregnancy and delivery (e.g., effects & type of placentation, intrauterine hypoxia, fetal transfusion syndrome, timing of the cleavage of the zygote (Derom & Thiery, 1976; Corey, Nance, Kang, & Christian, 1979; Bjoro & Bjoro, 1985; O’Brian & Hay, 1987). Corey et al. (1979) suggest a possible maternal effect on fetal growth. If the intrauterine environment is deficient in some way, one twin may be forced to compete for nutrients at the expense of the co-twin, resulting in very different intrauterine experiences for each twin. Another environmental difference called the fetal transfusion syndrome has been discovered in monochorionic monozygotic twins. In these twins, an intrauterine blood transfer can occur from one twin (the donor) to the other (the recipient). This vascular interconnection during fetal life has been found to cause an imbalance of blood between the fetuses resulting in large intrapair birthweight differences in these twins. This difference between members of a monochorionic twin pair have also been related to cognitive differences observed at a later age with the lighter twin having poorer nonverbal scores if he is male (O’Brian & Hay, 1987). In addition to the transfusion syndrome, other prenatal differences between members of a twin pair have been studied (e.g., Derom & Thiery, 1976; Bjoro & Bjoro, 1985). Derom & Thiery (1976) studied anaerobic metabolism and cord blood acid-base balance differences between members of a twin pair. This phenomenon called intrauterine hypoxia is peculiar only to the second-born twin. Bjoro & Bjoro (1985) investigated several factors thought to retard intrauterine growth in twins and found great differences between members of a twin pair particularly for birthweight, and hypothesized that insufficiencies in the maternal supply line, marginally and velamentously inserted cords, and placental function could cause this difference. Intrapair weight differences were also found to be greatest in twins with fused dichorionic placentae than in those with separate ones, although there were no differences between the intrapair variations of MZ and DZ twins. These studies all give evidence for the kinds and extent of noninherited factors that are not shared by members of a twin pair and their effects on fetal growth and development. Several statements can now be made. First, research has demonstrated that individual differences in sex steroids at birth are related to some sexually dimorphic behaviors (Jacklin et al., 1984; Jacklin et al., 1983; Jacklin et al., 1988). Second, this study has demonstrated that variations of neonatal levels of estradiol and progesterone are mediated by genetic factors. Possibly then, the relationships between hormones and sexually dimorphic behaviors are due, in part, to inherited genes. In fact, Mitchell, Baker, & Jacklin (1989)reported evidence for genetic factors influencing individual differences in both masculine and feminine personality traits in 9- to 13-year-old children. Finally, the prenatal experiences of the fetus appear to be an important factor in the variation and covariation of sex steroids observed at birth. Significant relationships have been demonstrated between these same steroids and later behaviors (e.g., Jacklin et al., 1988), suggesting that even prenatal experiences may play a role in producing long-term behavioral differences between twins. Although several sources of shared and nonshared environmental influences have been identified, a vast array of other parity-related factors have yet to be seriously considered (e.g., maternal age, birth order, length of labor). This further emphasizes the importance of the maternal role in the growth and development of the child. A mother’s experiences during her pregnancy have an effect on the prenatal experiences of the child, and these
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prenatal experiences may have an important influence not only on neonatal sexsteroid levels, but on later behaviors. Hormone-behavior relationships are complex. Genes appear to influence some hormone levels, but these genes are not immutable and their roles are not fixed. Genes can interact with environmental experiences, and both these factors influence behavior. Prenatal care and experiences also play a role in the variation and covariation of neonatal sex-steroid levels. Further research is needed to help identify the exact nature of these influences, and the interrelationships between maternal effects, unique experiences, hormones, and even behavior observed at a later age.
Notes This project was supported by a Faculty Research and Innovation Fund grant to the second and third authors at the University of Southern California. We are grateful to numerous research assistants involved in this project, especially Lina Babani, Jilla Carpey, Elvira Garcia, and Mary Villanueva. Special thanks to the staff of the USC/Los Angeles County Medical Center for their time and effort.
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Maccoby, E. E., & Jacklin, C. N. (1974). Myth;reality, and shades of gray: What we know and don’t know about sex differences. Psychology Today, 8, 109-112. Marcus, J., Maccoby, E. E., Jacklin, C. N., & Doering, C. H. (1985). Individual differences in mood in early childhood: Their relation to gender and neonatal sex steroids. Developmental Psychobiology, 18, 327-340. Maynard, P. V., Heyes, V. M., & Shaxted, E. J. (1982). Plasma progesterone in the umbilical vessels at vaginal delivery of term infants. British Journal of Obstetrics and Gynaecology, 89, 989-993. Meisel, R. L., & Ward, I. L. (1981). Fetal female rats are masculinized by male littermates located caudally in the uterus. Science, 213, 239-242. Mitchell, J. E., Baker, L. A., & Jacklin, C. N. (1989). Masculinity and femininity in twin children: Genetic and environmental factors. Child Development, 60, 1475-1485. O’Brian, P. J., & Hay, D. A. (1987). Birthweight differences, the transfusion syndrome, and the cognitive development of monozygotic twins. Acta Genet. Med. Gemellol., 36, 181-196. Shutt, D. A., Smith, I. D., & Shearman, R. P. (1974). Fetal plasma steroids in relation to paturation: 111. The effects of panty and method of delivery upon umbilical plasma oestrone and oestradiol. The Journal of Obstetrics and Gynaecology of the British Commonweallh, 81, 968-970. Tobet, S. A , , Dunlap, J. L., & Gerall, A. A. (1982). Influence of fetal position on neonatal androgeninfluenced sterility and sexual behavior in female rats. Hormones and Beahuior. 16, 251-258. Vlietinck, R., Derom, R., Neale, M. C., Maes, H., van Loon, H., Derom, C., & Thiery, M. (1989). Genetic and environmental variation in the birthweight of twins. Behavior Genetics, 19, 151-161. Wilson, J. D., & Foster, D. W. (1985). Textbook ofendocrinology. Philadelphia, PA: W. B. Saunders Company. Wilson, J. D., George, F. W., & Griffin, J. E. (1981). The hormonal control of sexual development. Science, 211, 1278-1284.
IRA SHULMAN Los Angeles CountylUSC Medical Center Los Angeles, California
Three sex-steroids (estradiol, progesterone, & testosterone) were assayed from the umbilical cord blood of 58 same-sex twin pairs in an investigation of the effects of sex, as well as genetic and environmental factors, on neonatal hormone levels. Although significant mean differences were found between boys and girls for both testosterone and progesterone, sex appeared to account for very little of the total variation for any of the hormones. Results showed that genetic influences significantly affected within-sex variation in both estradiol and progesterone levels, while variations in the intrauterine (shared twin) environment accounted primarily for differences in levels of testosterone. Moderate correlations were also found among the three hormones. Multivariate biometrical analyses revealed these relationships to be explained by an underlying general factor of nonshared environmental influences affecting all three hormones. Genetic factors appeared to be specific to each hormone rather than correlated across hormones. These results suggest not only that genes are operating at this early age, but also that maternal and other prenatal factors (e.g., placental effects, uterine position) have a significant role in variations of sex-steroids and possibly on later behaviors.
Psychologists have recently become very interested in the individual differences in sex-steroid hormones and their role in anatomical, functional, and behavioral development. Although some sex differences in certain hormones do exist, which may in turn relate to some sexually dimorphic characteristics, there is also considerable variation within males and females for the same sex-steroid hormones (e.g., Marcus, Maccoby, Jacklin, & Doering, 1985; Jacklin, Maccoby, Doering, & Reprint requests should be sent to Laura A. Baker, Department of Psychology, SGM 501, Los Angeles, CA 90089-1061, U.S.A. Received for publication 1 1 December 1989 Revised for publication 14 August 1991 Accepted at Wiley 9 September 1991 Developmental Psychobiology 24(8):559-570 (1992) 0 1992 by John Wiley & Sons, Inc.
CCC 00 12-1630/92/080SS9- 12$04.00
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King, 1984; Maccoby, Doering, Jacklin, & Kraemer, 1979). To date, however, the causes of these individual differences and the mechanisms underlying their relationships to psychological and physical characteristics remain unknown. Both the nervous system and the endocrine system are recognized as control mechanisms that regulate hormone behavior relationships. This control begins early during fetal development. The human brain grows most rapidly between the middle of gestation through the second year after birth. During this development, there are critical periods during which the brain is most susceptible to the presence of particular hormones. Gonadal hormones can imprint a functional pattern on the brain during this critical period which influences future patterns of behavior. For example, brain development begins as fundamentally female. However, the presence of androgen produced by the testis will impose a masculine pattern of development on the fetus. Sex hormones can thus play an organizing role on the physical development of the fetus and are quite influential even prenatally. The organizing effects of hormones on human development has been generalized to include hormonal influences on individual differences in cognitive abilities and sexually dimorphic behaviors (Wilson & Foster, 1985). Sex steroids are important determinants of certain differences between males and females, such as genitalia differentiation (Wilson, George, & Griffin, 198 l), brain organization, and sexual behavior (see Hines, 1982). In addition, the nature of sex differences may change at different times in the life cycle. That is, sexsteroid levels of men and women differ markedly during puberty, but less overlap may be seen in early fetal life. In recent years, however, it has become increasingly evident that there are substantial variations of these same hormones within each sex. It is no longer viewed that there are exclusive male or female hormones, per se, but that both sexes possess the same hormones, albeit in varying degrees. Sexual differentiation is rather the result of differences in levels of individual hormones in males and females, rather than the presence or absence of certain hormones (Wilson & Foster, 1985). These within-sex hormonal variations have also been associated with individual differences in cognitive performance and social behavioral development. Most surprisingly, neonatal sex hormones appear to be the chemical mediators of some sexually dimorphic human behaviors displayed at later ages. For example, individual differences in testosterone and androstenedione in umbilical cord blood have been reported to be significant predictors of spatial ability in 6-year-old girls, while timidity scores in 6- to 18-month-old boys were related to neonatal levels of progesterone, testosterone, and estradiol (Jacklin, Maccoby, & Doering, 1983; Jacklin, Thompson-Wilcox, & Maccoby, 1988). These provocative findings suggest that patterns of stereotypical masculine or feminine behaviors may be partly biologically based rather than solely a result of social learning. However, it must be emphasized that within-sex variations appear far greater than between-sex differences in sex hormones (Maccoby, et al., 1979; Hines, 1982) as well as in most sexually dimorphic behaviors (Maccoby & Jacklin, 1974). Thus, an important step in our understanding of hormone-behavior relationships must be to realize the sources of these individual differences in sex hormones in males and females. While previous research has implicated the role of circulating maternal hormones and parity factors in neonatal sex-steroid levels (e.g., Maynard, Heyes, & Shaxted, 1982; Shutt, Smith, & Shearman, 1974), these only account
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for a small portion of variation of hormone levels at birth. The importance of other characteristics of the uterine environment, as well as of the fetus itself, remain important questions in our understanding of both within- and between-sex differences in sex-hormone levels. Genetic variations among individuals are included in the most important classes of phenotypic or observed variance in most behavioral and physical characteristics. However, the role of genetic factors in individual differences in sexsteroid levels has yet to be studied. The twin design provides a basis for which questions concerning fetal genetic, uterine environment, and other maternal factors may be addressed. Hormonal assays were performed on umbilical cord blood at birth for 36 monozygotic (MZ) and 22 dizygotic (DZ) twin pairs. Although these cord-blood samples are reflective of maternal, fetal, and placental units, these various effects may be considered in a genetic and environmental framework of individual differences. For example, maternal effects should produce similarities between co-twins, regardless of their zygosity (MZ or DZ), thus reflecting shared environmental influences. On the other hand, to the extent that placental units produce differences between co-twins, these would be represented as unique or nonshared environmental effects. Finally, genetic variations in fetal units will produce less observed similarities in DZ twin pairs, who share only half their nonsegregating genes, as compared to the genetically identical MZ twins. In addition to parsing hormonal variation into genetic, shared environmental, and nonshared environmental sources, the twin design allows genetic and environmental covariation among several hormones to be investigated through multivariate biometrical analyses. Such analyses are necessary in the present study since neonatal levels of these sex steroids have been previously shown to be positively correlated, indicating a lack of independence among them (Marcus et al., 1985). For example, it is recognized that testosterone serves as a substrate for estradiol. The observed correlation between these steroid levels at birth may be due to correlated genetic influences, correlated environmental influences, or both. A genetic correlation may relfect linkage disequilibrium and/or pleiotropic (multiple) effects of the same genes on the two hormones. Similarly, environmental correlations among the hormones would point to some aspect(s) of the maternal environment which influence levels of two or more sex steroids.
Method Design This study used a multivariate twin design to investigate inherited (genetic) and noninherited (environmental) influences on levels of estradiol, progesterone, and testosterone in umbilical cord blood samples taken at birth. Radioimmunoassays were performed on plasma samples of both monozygotic (MZ) and dizygotic (DZ) twins. The similarity between twins for each of the three hormones was then assessed. The twin design is based on the knowledge that MZ twins share 100% of their genes while DZ twins share, on the average, 50% of their segregating genes. If genetic factors influence a given trait, such as the level of estradiol, MZ twin pairs are expected to demonstrate significantly greater phenotypic similarity as
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compared to DZ pairs. Intraclass correlations may be calculated separately for MZ and DZ twins using within- and between-pair mean squares (MSW, MSB) as follows:
'
MSB - MSW = MSB + MSW
Heritability (h2),or the proportion of observed (phenotypic) variance explained by genetic factors, can be roughly estimated by doubling the difference between the within-pair correlation for.MZ twins and that for DZ twins (Falconer, 1981). That is,
h2 = 2(rMz - rDz). Proportions of variance explained by environmental factors common to members of the same twin pair ( c 2 )and by specific environmental factors unique to each individual (s *), can also be obtained algebraically from these zero-order, isomorphic correlations (rMzand rDZ). In multivariate analyses presented in this article, of additional interest are the cross-correlations between a given trait in one twin, such as his or her estradiol level, with a different trait in the co-twin, such as the level of testosterone. If genetic factors for estradiol are correlated with genetic factors for testosterone, this cross correlation is expected to be of greater magnitude in MZ twin pairs than in DZ pairs. However, if correlated environmental influences are the primary source of covariation between estradiol and testosterone levels, then cross-correlations should be comparable for MZ and DZ pairs. The extent to which correlated genetic and correlated environmental influences contribute to the phenotypic correlation between two traits, X and Y , may be readily seen in the following equation:
where hx and h, are square roots of heritabilities for traits X and Y respectively, cx and cy are square roots of common environmentalities for the two traits, and sx and sy are square roots of specific environmentalities. The cross-correlation bewhile rCXyand tween genetic influences for the two traits is symbolized by rGxyr rsxyrepresent the cross-correlations between environmental influences common to the two twins and between environmental influences specific to each twin. The LISREL-VI program (Joreskog & Sorbom, 1983) was used to estimate all of these genetic and environmental parameters simultaneously, as described by Fulker, Baker, and Bock (1984) and Ho, Baker, and Decker (1988). The procedure requires input matrices of between- and within-pair mean squares and cross products among the observed variables and yields constrained maximum-likelihood estimates of heritabilities, environmentalities, genetic correlations, and environmental correlations as described above. That is, observed variances and covariances are decomposed into diagonal matrices of square roots of heritabilities [h], common environmentalities [cl, and specific environmentalities [s] . Additional
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563
matrices contain genetic correlations [RG], common environmental correlations I&], and specific environmental correlations [R,]. Results from biometrical analyses will be presented in this more efficient matrix notation. Various models testing the relative influence of genetic and environmental sources of variance and covariance will be presented along with goodness-of-fit statistics.
Subjects The subjects consisted of 58 same-sex twin pairs (i.e., 116 infants) born at the Women’s Hospital on the USC Health Sciences campus. This hospital primarily serves women of low income as well as families on welfare. Twins included in this study primarily were of Hispanic descent and in good health. For the majority of twin pairs (96%), the gestational age exceeded 34.5 weeks and the birthweight of each co-twin exceeded 2250 g. These criteria are well within the normal range in the population of twin births (Vlietinck et al., 1989). Therefore, although the population might be considered at risk because of their poverty status, we sampled twins with an average birthweight that is considered high for twins in attempt to screen out other possible health complications. Birthweight was not found to be significantly related to hormone levels. Gestational age was significantly related only to levels of testosterone with older babies having more testosterone detected in their umbilical cord blood (r = .32, p < .05). Zygosity (MZ or DZ) was determined through antigen typing at the blood bank at the Los Angeles County/USC Hospital. For each twin, the presence or absence of 18 different antigens (A, B, D, C, E, C, G , M, N, K, E, Kpb, Fy”, Fyb, Jk”, Jkb, s, s), was determined. If a twin pair did not show an exact antigen match, they were considered DZ. On the other hand, twin pairs that showed an exact match of all 18 antigens were considered MZ. Although there is a remote chance that some of the pairs classified as MZ could conceivably be DZ, such misclassifications would not seriously affect our results. This procedure resulted in classification of 36 MZ and 22 DZ twin pairs.
Measures and Procedures Recent mothers of twins were reached through the Los Angeles County Women’s Hospital and informed consent was obtained to perform the red blood cell antigen-typings and sex-steroid radioimmunoassays on their twins. At the time of birth, 7-10 ml of umbilical cord blood was obtained for each twin and allowed to clot in a refrigerator. The serum was separated and then frozen at -20°C. Radioimmunoassays were subsequently performed on the frozen serum samples for three sex steroids: estradiol, progesterone, and testosterone. Progesterone kits were used to Extraction lZsIEstradiol and extraction 1251 measure levels of estradiol and progesterone respectively. These kits were purchased from Pantex in Santa Monica, California. The lowest detectable weight for estradiol was 10 pg/ml in serum. Intraassay coefficient of variation was 8% and interassay coefficient of variation was 10.5%. Sensitivity for progesterone was .5 ng/ml. Intraassay coefficient of variation was 7.5% and the interassay coefficient of variation was 10%. Levels of testosterone were determined with a Testosterone Radioimmunoassay (RIA) kit from the Diagnostic Products Corporation in Los
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Table 1 Means (SD) c$ Hormone Levels (nglml) by Gender and Zygosity Male Infants
Estradiol Progesterone* Testasterone*
Female Infants
MZ
DZ
( n = 46)
(n = 20)
9.15 (4.54) 621.23 (267.52) .69 ( .24)
10.33 (3.78) 678.90 (320.22) .82 (.19)
MZ (n = 26)
( n = 24)
DZ
8.15 (4.63) 465.73 (291.22) .55 (.I91
10.11 (6.91) 585.54 (264.98) .58 (.20)
“Indicates significant difference between males and females ( p 5 .05).
Angeles, California. Sensitivity for testosterone was 10 pg/dl and the intraassay coefficient of variation was 9.7%. Specificities for the various tests can be obtained from Pantex and Diagnostic Products Corporations. Assays were performed at the LA County/USC Endocrinology Lab twice for each sample to assess test-retest reliability.
Results Descriptive Statistics Hormone levels were calculated twice for each twin and test-retest reliability was then assessed for each hormone. Reliability coefficients were extremely high for all three hormones: testosterone ( r = .95), progesterone (r = .96), estradiol ( r = .99). Thus, individual hormone levels were calculated as the average level based on two separate assays. Mean hormone levels are reported by gender and zygosity in Table I . Means levels were consistently higher for DZ twins than for MZ twins although these differences were not statistically significant. Progesterone and testosterone levels were significantly higher for boy neonates than girl neonates ( p < .05). There were no significant effects of Zygosity nor any Sex x Zygosity interactions. Since there were significant differences between boys and girls for levels of both progesterone and testosterone, these sex effects were statistically removed from raw scores of each hormone using a multiple regression procedure to eliminate any spurious associations among the steroids due to their shared sex variation. Residual scores from the multiple regression of each steroid on sex were next computed. These residual scores were then transformed into standardized z scores with zero means and unit variances. All subsequent analyses presented here are based on sex-adjusted scores. As expected, there were moderate but significant correlations among the three sex-steroid levels (see Table 2), with correlations similar in magnitude and direction to those reported by Marcus and colleagues (1985). These interhormone relationships, or lack thereof, may be understood in part by what is currently known about the chemical processes involved in hormone production. That is, since testosterone
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Table 2 Correlations among Sex Steroids Estradiol Estradiol Progesterone Testosterone
1 .o .31* .41*
Progesterone
Testosterone
1 .o .11m
1 .o
*p < .0001 < .lo.
"p
serves as a substrate for estradiol, a correlation between levels of these hormones is expected whereas no correlation might be expected between testosterone and progesterone since they do not serve as a substrate for one another in humans. However, what is of interest is not simply whether these relationships exist but rather the underlying etiologies of these relationships. For example, are levels of testosterone correlated with levels of estradiol because the same sets of genes are influencing both steroids or are different genes at work?
Biometrical Analyses In order to investigate inherited and noninherited influences in the variation and covariation of each of these three hormone levels, multivariate biometrical analyses were performed. Several models were fit to 3 x 3 between- and withinpair mean squares and cross-product matrices among levels of estradiol, progesterone, and testosterone, computed separately for MZ and DZ twin pairs. Goodnessof-fit statistics for the various models are summarized in Table 3. First, a full model containing matrices [ h l , [cl, [sl, [&I, [ R , ] , and [ R , ] was fit to the data (Model I). This allowed both inherited and noninherited influences as well as correlations among the hormones within each class of influences to be estimated. The full model yielded a nonsignificant chi square, ~ ~ ( 1 = 2 )8.82, p = .18, indicating that this model with correlated genetic and correlated environmental factors fit the data quite well. Models constraining one or more of the estimated parameters to be zero were next fit to the data. These constrained models allowed us to test the relative importance of inherited versus noninherited influences. First, [ h ] and [R,] matrices were constrained to be a zero matrix. That is, in this model, genetic influences were set to zero and any twin similarity must thus be accounted for by common environmental variance. Test of this model yielded a significant chi square, ~ ~ ( 1 2 ) Table 3 Biometrical Analyses of Estradiol, Progesterone, and Testosterone Model
x2
df
P
1. Full model 2. [hl = [RG] = 0 3. [c] = [R,] = 0 4. Final model
8.82 24.81 26.63 19.20
6 12 12
.18
15
.20
.02 .01
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Table 4 Maximum-Likelihood Parameter Estimates from Final Model Estradiol
Progesterone .72*
.68*
0
0
.32*
Testosterone 0
.46* .28*
.54*
1.oo
.62* *p
.31*
1 .OO
1
< .05.
= 24.81 ,p < .02, with a significant increase compared to the full model, x2difference (6) = 1 5 . 9 9 , < ~ .05, and an overall poor fit to the data. Therefore, genetic variances and/or covariances may account for a proportion of the observed twin similarity and must be included in the final model. Dropping common environmental influences ([c] and [R,] matrices; Model 3) also produced apoor fit to the data, ~ ~ ( 1 = 2 26.63, ) p < .02, indicating that the shared environment factors also play a significant role in the variation andlor covariation of sex-steroid levels. Although the full model yielded a nonsignificant chi square and a good fit to the data, several parameters in this model were found to be nonsignificant upon closer examination. These included genetic correlations among the hormones as well as the genetic variance for testosterone. In addition, variations in the common environment were negligible and nonsignificant for all hormones except testosterone. Covariation among intrauterine effects (i.e., common environmental correlations) for the three hormones were also nonsignificant. Thus, these nonsignificant parameters were constrained to be zero in the final model (Model 4). This final model yielded a nonsignificant chi square, ~ ' ( 1 5 ) = 19.20, p = .21, and a nonsignificant difference from the full model, x2 difference (9) = 10.38, p > .IO. Parameter estimates based on this final model are presented in Table 4. Since correlations among genetic factors and among shared environmental factors were all found to be nonsignificant in these data, observed relationships among these three hormones are entirely explained by correlations among nonshared environmental effects (i.e., R,). Furthermore, estimates from matrix [R,] appear to be uniform and suggested an underlying general factor affecting all three sex steroids. In addition, specific environmentalities (s2), heritabilities (h2),and common environmentalities ( c 2 )for each hormone were computed. As seen in Table 4, there were significant genetic influences for both estradiol and progesterone, although genetic influences appeared to be specific to each hormone rather than correlated across hormones. Genetic factors did not seem to influence testosterone levels. Variability in testosterone was instead mediated by intrauterine factors that are common to twin pairs ( c 2 = .46). The remaining phenotypic variance for each hormone was explained by environmental factors not shared by twins. This may include, for example, differences in the position of each twin in the uterus or placentation effects.
Discussion The present investigation of newborn twins therefore provides the first attempt to determine the relative contributions of sex, environmental, and genetic factors
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to variations in neonatal levels of three sex steroids: estradiol, testosterone, and progesterone. Hormonal assays were performed on umbilical cord blood at birth. Although these cord-blood samples are reflective of maternal, fetal, and placental units, their various effects may be considered in a genetic and environmental framework of individual differences. By comparing the observed (phenotypic) resemblance of MZ and DZ twin pairs for levels of hormones, effects of genetic factors operating in the fetus were separated from other maternal environmental influences. Furthermore, the extent to which the prenatal environment produced similarities or differences between co-twins were investigated. That is, a distinction was made between aspects of the environment which were shared or common to the twins (e.g., circulating nutrients in the womb, maternal hormone levels), and those which were not shared or are unique to each twin (e.g., position in the uterus). Differences in hormone levels between males and females were in the expected directions with boys having higher levels of testosterone and progesterone than girls. However, unlike other times during the life cycle (e.g., puberty), sex differences accounted for very little of the total variation of any of these neonatal hormones ( r 2 = .001, .037, and .129 for estradiol, progesterone, & testosterone, respectively). As is often found in studies of sex differences in behavior, there was substantially greater within-sex variation in sex hormones compared to betweensex differences. In addition, these three sex steroids were moderately intercorrelated. Environmental factors specific to each twin appeared to account for this covariation. Also note that these specific or nonshared environmental factors contributed significantly to individual differences in levels of each specific hormone. The significant role of these specific environmental factors in the variation and covariation of sex steroids was particularly interesting, especially since both MZ and DZ co-twins share the same uterine environments at the same time. In spite of this, there were apparently enough differences within a given pregnancy to account for substantial variation between the twins. These factors could be prenatal experiences unique to each twin such as position in the uterus and other placentation effects as discussed further below. Moreover, whatever the source of these within-uterus differences, they appeared to influence all three hormones simultaneously. Several environmental factors may be unique to each twin. One possibility comes from neuroendocrinology studies on nonhuman animals. Studies on rats give evidence that position in the uterus may influence the presence of androgens in littermates. Male rats located on the caudal side of the females appear to have a masculinizing effect, thus influencing the birth weight, mounting behavior, and responsiveness to the activational effects of testosterone (Babine & Smotherman, 1984; Meisel & Ward, 1981). The masculinizing effects of intrauterine, contiguous male littermates on female behavior have also been demonstrated with guinea pigs, mice, and hamsters (Gandelman, 1986; Kinsley et al., 1986; Tobet, Dunlap, & Gerall, 1982). Since this effect has been observed only in nonhuman animal research, it is difficult to directly compare this information to our study on same-sex human twins, and extreme caution must be taken when generalizing these effects. However, it does suggest that possible differences occur in what is typically viewed as a rather homogeneous uterine environment. That each individual twin has unique experiences within the womb appears highly tenable. Other evidence for prenatal differences between members of a twin pair come
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from studies on pregnancy and delivery (e.g., effects & type of placentation, intrauterine hypoxia, fetal transfusion syndrome, timing of the cleavage of the zygote (Derom & Thiery, 1976; Corey, Nance, Kang, & Christian, 1979; Bjoro & Bjoro, 1985; O’Brian & Hay, 1987). Corey et al. (1979) suggest a possible maternal effect on fetal growth. If the intrauterine environment is deficient in some way, one twin may be forced to compete for nutrients at the expense of the co-twin, resulting in very different intrauterine experiences for each twin. Another environmental difference called the fetal transfusion syndrome has been discovered in monochorionic monozygotic twins. In these twins, an intrauterine blood transfer can occur from one twin (the donor) to the other (the recipient). This vascular interconnection during fetal life has been found to cause an imbalance of blood between the fetuses resulting in large intrapair birthweight differences in these twins. This difference between members of a monochorionic twin pair have also been related to cognitive differences observed at a later age with the lighter twin having poorer nonverbal scores if he is male (O’Brian & Hay, 1987). In addition to the transfusion syndrome, other prenatal differences between members of a twin pair have been studied (e.g., Derom & Thiery, 1976; Bjoro & Bjoro, 1985). Derom & Thiery (1976) studied anaerobic metabolism and cord blood acid-base balance differences between members of a twin pair. This phenomenon called intrauterine hypoxia is peculiar only to the second-born twin. Bjoro & Bjoro (1985) investigated several factors thought to retard intrauterine growth in twins and found great differences between members of a twin pair particularly for birthweight, and hypothesized that insufficiencies in the maternal supply line, marginally and velamentously inserted cords, and placental function could cause this difference. Intrapair weight differences were also found to be greatest in twins with fused dichorionic placentae than in those with separate ones, although there were no differences between the intrapair variations of MZ and DZ twins. These studies all give evidence for the kinds and extent of noninherited factors that are not shared by members of a twin pair and their effects on fetal growth and development. Several statements can now be made. First, research has demonstrated that individual differences in sex steroids at birth are related to some sexually dimorphic behaviors (Jacklin et al., 1984; Jacklin et al., 1983; Jacklin et al., 1988). Second, this study has demonstrated that variations of neonatal levels of estradiol and progesterone are mediated by genetic factors. Possibly then, the relationships between hormones and sexually dimorphic behaviors are due, in part, to inherited genes. In fact, Mitchell, Baker, & Jacklin (1989)reported evidence for genetic factors influencing individual differences in both masculine and feminine personality traits in 9- to 13-year-old children. Finally, the prenatal experiences of the fetus appear to be an important factor in the variation and covariation of sex steroids observed at birth. Significant relationships have been demonstrated between these same steroids and later behaviors (e.g., Jacklin et al., 1988), suggesting that even prenatal experiences may play a role in producing long-term behavioral differences between twins. Although several sources of shared and nonshared environmental influences have been identified, a vast array of other parity-related factors have yet to be seriously considered (e.g., maternal age, birth order, length of labor). This further emphasizes the importance of the maternal role in the growth and development of the child. A mother’s experiences during her pregnancy have an effect on the prenatal experiences of the child, and these
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prenatal experiences may have an important influence not only on neonatal sexsteroid levels, but on later behaviors. Hormone-behavior relationships are complex. Genes appear to influence some hormone levels, but these genes are not immutable and their roles are not fixed. Genes can interact with environmental experiences, and both these factors influence behavior. Prenatal care and experiences also play a role in the variation and covariation of neonatal sex-steroid levels. Further research is needed to help identify the exact nature of these influences, and the interrelationships between maternal effects, unique experiences, hormones, and even behavior observed at a later age.
Notes This project was supported by a Faculty Research and Innovation Fund grant to the second and third authors at the University of Southern California. We are grateful to numerous research assistants involved in this project, especially Lina Babani, Jilla Carpey, Elvira Garcia, and Mary Villanueva. Special thanks to the staff of the USC/Los Angeles County Medical Center for their time and effort.
References Babine, A. M., & Smotherman, W. P. (1984). Uterine position and conditioned taste aversion. Behauioral Neuroscience, 98, 461-466. Bjoro, K., Jr., & Bjoro, K. (1985). Disturbed intrauterine growth in twins: Etiological aspects. Acta Genetical Medicae et Gemellogicae, 34, 73-79. Corey, L. A., Nance, W. E., Kang, K. W., & Christian, J. C. (1979). Effects of type of placentation on birthweight and its variability in monozygotic and dizygotic twins. Acru Genetical Medicae et Gemellogicae, 28, 41-50. Derom, R., & Thiery, M. (1976). Intrauterine hypoxia-A phenomenon peculiar to the second twin. Acta Genetical Medicae et Gemellogicae, 25, 314-316. Falconer, D. S. (1981). Introduction to quantitative genetics (2nd e d . ) . London: Longman Group. Fulker, D. W., Baker, L. A., & Bock, R. D. (1984). Estimating components of covariance using LISREL. Data Analyst, 1, 5-8. Gandelman, R. (1986). Uterine position and the activation of male sexual activity in testosterone proprionate-treated female guinea pigs. Hormones and Behavior, 20, 287-293. Hines, M. (1982). Prenatal gonadal hormones and sex differences in human behavior. Psychological Bulletin, 92, 56-80. Ho, H-Z., Baker, L. A., & Decker, S. N. (1988). Covariation among intelligence and cognitive processing rates: Genetic and environmental influences. Behavior Genetics, 18, 247-277. Jacklin, C. N., Maccoby, E. E., & Doering, C. H. (1983). Neonatal sex-steroid hormones and timidity in 6- to 18-month-old boys and girls. Developmental Psychobiology, 16, 163-168. Jacklin, C. N., Maccoby, E. E., Doering, C. H., &King, D. R. (1984). Neonatal sex-steroid hormones and muscular strength of boys and girls in the first three years. Developmental Psychobiology, 17, 301-310. Jacklin, C. N., Thompson-Wilcox, K., & Maccoby, E. E. (1988). Neonatal sex-steroid hormones and cognitive abilities at six years. Developmental Psychobiology, 21, 567-574. Joreskog, K . , & Sorbom, D. (1983). LISREL: Analysis of linear structural relationships by the method of maximum-likelihood (Version Vf).Chicago: International Educational Services. Kinsley, C., Miele, J., Konen-Wagner, C., Ghiraldi, L., Broida, J., & Svare, B. (1986). Prior intrauterine position influences body weight in male and female mice. Hormones andBehavior,20,201-21 1. Maccoby, E. E., Doering, C. H., Jacklin, C. N., & Kraemer, H. (1979). Concentrationsofsex hormones in umbilical cord blood: Their relation to sex and birth order of infants. Child Developmenr, 50, 632-642.
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