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Original Studies |
Department of Behavioral and Social Sciences, Southern Illinois University School of Medicine (S.A.B., K.B.), Carbondale, Illinois 62901; and the Department of Pediatrics, Northwestern University School of Medicine and Evanston Hospital (S.C.D.), Evanston, Illinois 60201
Address all correspondence and requests for reprints to: Dr. Sheri A. Berenbaum, Department of Behavioral and Social Sciences, Southern Illinois University School of Medicine, Carbondale, Illinois 62901. E-mail: sberenbaum{at}som.siu.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The behavioral differences observed between females with CAH and unaffected females are generally considered to reflect the high levels of androgen present early in development before the disorder is detected and treatment initiated. In fact, females with CAH were initially studied to confirm findings in rodents and primates implicating early androgens in the development of sexually dimorphic behavior (18). Those studies showed that behavioral sexual differentiation is affected by the levels of gonadal hormones present early in development producing permanent changes in the structure of the brain and in its sensitivity to later activational effects of hormones (19, 20, 21). In humans, the prenatal period is considered the crucial time for sexual differentiation of the brain and behavior, because sexual differentiation of the genitalia and sex differences in hormone concentrations are greatest during weeks 8–24 of gestation (22, 23). Thus, the behavioral changes seen in females with CAH suggest that high levels of androgens specifically during prenatal life facilitate the development of male-typical behaviors and inhibit the development of female-typical behaviors (3, 24).
It is possible, however, that some of the behavioral changes in females with CAH reflect effects of elevated androgens in postnatal life. Individuals with CAH continue to have elevated androgen levels until and often after they are diagnosed and treated, and studies in other species indicate that hormonal effects on behavior are not confined to early development. Hormones may continue to affect behavior through permanent changes to the brain during early postnatal development (25, 26) and at later periods (27), but are more likely to activate neural systems that were organized early in life (19, 20, 21, 28).
There is only indirect evidence that the behavioral changes observed in CAH females result specifically from elevated androgen levels during prenatal development. First, studies in other species have shown that most aspects of behavioral sexual differentiation are affected more by the levels of hormones present during early development when the brain is developing than by hormones present later in development, although the expression of sexual behavior also requires the presence of appropriate hormones later in life (19, 20, 21). However, there is increasing recognition of the role of later hormones in both human and nonhuman behavior, most notably in aspects of learning, memory, and cognition (28, 29) and aggressive behavior (30). Second, most patients with CAH are vigorously treated to normalize postnatal androgens (31, 32), and, in fact, it may be more common for females to have subnormal levels of androgen than excess levels (33). Masculinized behavior of females with CAH has not been found to relate to the quality of therapy or aspects of disease control that should reflect postnatal androgen levels (6).
Nevertheless, there have been no systematic studies of the relative contributions of excess androgens at different points in development to behavioral masculinization and defeminization. Therefore, we attempted to study in females with CAH the relative contributions to behavior of androgen excess at different times, including the prenatal, early postnatal (first year of life), and later postnatal (childhood) periods. On the basis of animal studies and suggestive evidence in humans, childhood play behaviors were hypothesized to relate to androgen excess in the prenatal period and perhaps in the early postnatal period, whereas aggression was hypothesized to relate to androgen excess at all times.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The girls whose data are reported here are part of an ongoing longitudinal behavioral study of children with CAH and their siblings. Boys and girls with 21-hydroxylase-deficient CAH were recruited through university-affiliated pediatric endocrinology clinics in the midwestern United States. They represented a range of socioeconomic backgrounds, and almost all were Caucasian. Siblings and first cousins, similar in age, were recruited as controls. Assessments were conducted at several points in time on a variety of sex-typed behaviors. The research was approved by institutional review boards at all cooperating institutions. Parents provided informed written consent for behavioral assessments and evaluation of medical records, and children provided oral assent for behavioral procedures.
Details of the method and descriptions of the differences between children with CAH and their unaffected siblings have been reported previously (4, 5, 8, 9, 13, 34; see Ref. 2 for a summary). We focused here on timing of exposure to androgen excess in relation to childhood assessments of sex-typed play and activities, aggression, and interest in infants in 23 girls with CAH between the ages of 3–12 yr (mean, 8.3 yr).
Behavioral measures
Childhood boy-typical play was measured in several ways on two occasions, 6 months apart (5). In brief, 1) children played with a standard set of sex-typed toys for 10 min, and the amounts of time they played with boys’ toys, girls’ toys, and neutral toys were recorded; 2) children chose a sex-typed toy to keep, with the choice scored as boy-typical or girl-typical; 3) children provided oral answers to questions about their preferences for a variety of sex-typed activities, and responses were scored for interest in boy-typical and girl-typical activities; and 4) parents provided written answers to questions about the child’s typical preferences for the same sex-typed activities, which were similarly scored for the child’s interest in boy-typical and girl-typical activities. At each session, a composite of boy-typical interests was derived from the four individual measures. As reported previously (5), girls with CAH differed significantly from their unaffected sisters on all measures, by showing greater interest and participation in boy-typical activities and less interest and participation in girl-typical activities. The differences on the composite measures of boy-typical play were very large (1.6 and 1.4 SD for sessions 1 and 2, respectively), and there was little overlap in the averaged composite scores of girls with CAH and control girls.
Aggression was measured by parent report of the child’s likely response to six hypothetical situations involving conflict. The score was the number of times the parent said the child would use physical aggression. As reported previously (9), girls with CAH were reported to be more likely to use physical aggression than their sisters, but the difference was only moderate in size (the difference was 0.4 SD) and was not significant in this sample of children. On the same instrument, but with self-report, adolescent and adult women with CAH reported themselves to be significantly more likely to use physical aggression than did their sisters.
Interest in infants was measured by parent report on a multiitem questionnaire of the child’s involvement in playing and caring for infants. As reported previously (13), girls with CAH were reported by their parents to be significantly less interested in infants than their sisters; the difference was large, 0.7 SD. To facilitate comparisons across behavioral measures, scoring of this measure was reversed, so that high scores reflect low interest in infants. Thus, for all behavioral measures, high scores reflect "masculine responses."
Indicators of androgen excess
Retrospective evaluation of medical records was performed by a pediatric endocrinologist (S.C.D.). All patient identifiers were removed. Ratings were made after all behavioral assessments were completed and without knowledge of behavioral results.
Prenatal androgen exposure was indexed by three aspects of the disease also related to disease severity (35, 36). Type of CAH was classified as simple virilizing (SV-), mild salt wasting (SW-), or severe SW. SW-CAH required elevated PRA for age and responsiveness to mineralocorticoid therapy. Classification as mild SW-CAH required that all measurements of serum sodium be at least 129 mmol/L, and that there be no episodes of hypotension or shock; classification as severe SW-CAH required that serum sodium in the neonatal period be less than 129 mmol/L, or that there be at least one episode of hypotension or shock. Age at diagnosis was classified as neonatal (<6 months), infancy (6–36 months), or childhood (>36 months). Prader ratings (37) were used to describe masculinization of the external genitalia at diagnosis.
Early postnatal androgen exposure was measured by growth and concentrations of 17-hydroxyprogesterone (17-OHP). Growth rate in the first year of life was considered the best measure, because advancing growth reflects the continuing effects of androgen excess. Height data were compared to National Center for Health Statistics norms to determine whether the child’s growth was normal (slope was parallel to the 50th percentile), decelerating, or accelerating. Concentrations of 17-OHP at the first neonatal assessment and at 6 months of age were used as supplementary indicators of androgen excess in the early postnatal period. Although large deviations generally reflect inadequate androgen suppression, a variety of factors (e.g. time of collection) can influence 17-OHP levels, reducing their reliability and validity as indicators of androgen. Levels of 17-OHP were recorded in the original units (nanograms per dL) and in log-transformed units to reduce the effects of extremely high values.
Later postnatal (childhood) androgen exposure was also measured with a
primary and supplementary indexes. Bone age advance was considered to
be the best measure, because it reflects a cumulative effect of excess
androgen. Annual radiographic evaluation of the bone age x-ray value
reported in the clinic note was noted. Bone age advance across
childhood was determined from change in bone age relative to the change
in chronological age (see Fig. 1
). Growth
rate and 17-OHP concentrations after the first year were used as
supplementary measures of later postnatal androgen excess. Growth rate
was recorded to be normal, decelerating, or accelerating (relative to
National Center for Health Statistics growth data) in the second
year, in early childhood (yr 2–5), and in middle childhood. The level
of 17-OHP closest in time to behavioral testing was recorded, as was a
typical 17-OHP level, based on 17-OHP values averaged across multiple
assessments in childhood (all 17-OHP values were recorded in original
and log-transformed units).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Most girls in this study generally had severe disease and so were
exposed to relatively high levels of androgens in utero
(Table 1). Indicators of prenatal
androgen excess were correlated with each other. Type of CAH (0,
SV-CAH; 1, mild SW-CAH; 2, severe SW-CAH) correlated significantly with
age at diagnosis (n = 23; r = -0.80; P <
0.001) and Prader score (n = 18; r = 0.54; P
< 0.05). Age at diagnosis correlated significantly with Prader score
(n = 18; r = -0.70; P < 0.01). The
correlations were attenuated when the two girls with SV-CAH were
excluded (CAH type and age at diagnosis: n = 21; r = -0.62;
P < 0.01; CAH type and Prader score: n = 16;
r = 0.29; P = NS; age at diagnosis and Prader
score: n = 16; r = -0.57; P < 0.05.) As
expected, girls who were diagnosed in the neonatal period had high
initial levels of 17-OHP, but their disease was brought quickly under
control, as reflected in 6 month levels of 17-OHP and growth rate in
the first year (Table 2). (One girl with
severe SW-CAH was treated with dexamethasone during the prenatal
period, but the treatment was only moderately successful, as reflected
by a Prader score of 3 and a neonatal 17-OHP of 20,800 ng/dL. The
results of all analyses were unchanged when she was excluded.)
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). Concentrations of 17-OHP varied over
time, partly due to the timing of specimen collection, patient
compliance, and time intervals between clinic visits. Medians were
substantially lower than means, reflecting the fact that most girls had
low levels, but a few girls had high levels. Cross-time stability of
17-OHP levels was assessed with Spearman rank-order correlations
(preserving the relative ordering of girls, but minimizing effects of
extreme values). The median correlation was 0.35, indicating that the
same girls did not have high levels each time.
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shows the relation of bone age to chronological age for
individuals girls. Bone age was appropriate for or lower than
chronological age for 10 of the15 girls for whom sufficient data were
available. Of the 5 girls with advanced bone age, 4 had mild forms of
CAH, were diagnosed later, and had mild genital masculinization. Associations between behavior and inferred androgen excess in girls
Associations between behavior and androgen excess were assessed
with Pearson correlations. Sex-atypical behavior was primarily
associated with indicators of prenatal androgen excess, not with later
postnatal androgen excess. Masculinization of toy play in particular
was associated with severe salt wasting, early age at diagnosis, and
high Prader scores (Table 4 and Fig. 2).
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and 6).
Elevated 17-OHP at 6 months of age was associated with high scores on
physical aggression in childhood, and growth acceleration beyond age 5
yr was associated with boy-typical play. Otherwise, masculinized
behavior was actually negatively associated with indicators of
postnatal androgen excess, most notably with bone age advance, the best
indicator. For example, girls with advanced bone ages actually had less
boy-typical play than girls with age-appropriate bone ages.
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Missing data
Although medical records were available for all girls, there was considerable variability in the amount of information that could be used from those records, primarily because of missed clinic visits and insufficient recording of laboratory results. We assessed the effect of this problem by comparing girls with a limited amount of data to those with more data. There was some suggestion that girls with little medical data were more behaviorally masculinized and had more severe illness than those with good data, but the small number of subjects made it difficult to draw firm conclusions.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our findings are novel in several ways. First, unlike others, we found that behavior was related to the degree of genital masculinization at diagnosis (4, 6, 13, 39). Discrepancies across studies may reflect statistical differences. In particular, the use of multiple measures increases reliability and ability to detect associations, as reflected in significant correlations with composite measures of boy-typical play, but not with single measures of aggression and interest in infants. Although it has been suggested that behavioral changes in females with CAH result from parent treatment in response to masculinized genitalia (40), our data suggest instead that both the behavior and the masculinized genitalia are caused directly by prenatal androgen. This is consistent both with parents’ reports that they did not treat CAH females in a masculine fashion (4, 7) and with data from androgenized female rhesus macaques that showed no relation between maternal behavior and androgen-influenced offspring behavior (41).
Second, we found indicators of prenatal androgen exposure to be most strongly associated with measures of childhood boy-typical play. Associations with aggression were inconsistent, and there were no significant positive associations with low interest in infants. This is compatible with the fact that childhood play activities show large and persistent differences between females with CAH and unaffected females (2) and between typical males and females (42, 43), whereas there are smaller differences in interest in infants and aggression (3, 9, 13). Of note, however, assessment of boy-typical play was probably more reliable than was assessment of aggression and interest in infants, and so the former were more likely than the latter to show associations with other measures. In this regard, it is interesting to note that the relation between physical aggression and Prader score was of moderate magnitude (r = 0.39) in the whole sample (although it was not significant given the sample size) and quite substantial (r = 0.69) in the subgroup of girls with severe SW-CAH. Therefore, it will be valuable to examine this issue further, using multiple measures of aggression and interest in infants.
Third, our results are inconsistent with the idea that masculinization of behavior results primarily from postnatal androgen excess. There were few significant correlations between behavior and indicators of postnatal androgen excess. Boy-typical play was associated with accelerated growth rate beyond age 5 yr, primarily due to two girls with accelerated growth and very high scores for boy-typical play. This growth probably reflected catch-up rather than advancing growth, because behavior was not positively related to growth rate at any other time, was not related to 17-OHP concentrations, and was negatively associated with bone age, a better indicator of growth advancement. In fact, boy-typical play and low interest in infants were associated with relatively low levels of postnatal androgen, probably because girls with severe illness had a greater degree of adrenal suppression than girls with mild illness. Further, postnatal androgen excess varied considerably across individuals and across time, but almost all girls with CAH (except those with mild illness) preferred boys’ toys at both times studied here and at other ages (4, 8).
Physical aggression in childhood was significantly associated with one indicator of early postnatal androgen excess, elevated 17-OHP at 6 months of age. Aggression was not significantly associated with other indicators of postnatal androgen, either early or in childhood. It is difficult to know whether the single significant correlation reflects the importance of the early postnatal period for the organization of brain systems subserving physical aggression or sampling fluctuation. In considering the few significant correlations with aggression and interest in infants, it is important to remember that our ability to examine relations between those behaviors and androgen excess was limited by the use of single measures.
We note several other limitations to our study. First, a considerable amount of medical information was missing. Although missing data may not be random, it is difficult to see how the loss of data from SW-CAH girls who were more behaviorally masculinized would produce the pattern of results obtained. Second, the relatively small sample has two effects. Statistical power is high enough to detect only large effects, so it is possible that postnatal androgens affect behavior in modest ways that could not be detected. Further, outliers may unduly influence results in small samples, so associations between behavior and indicators of prenatal androgen may be overestimated from this sample. Third, to increase statistical power, no corrections were made for multiple statistical comparisons. Although this increases the likelihood of type I error, it is justified given the dearth of information on the topic studied. Further, interpretations were based on consistency of results rather than isolated findings. Fourth, we were unable to examine behavior in relation to concurrent levels of androstenedione, which is a more direct index of androgen levels than is 17-OHP. Fifth, data were obtained from retrospective evaluation of medical records from several institutions. Although all ratings were made by a single pediatric endocrinologist from chart information provided by pediatric endocrinologists, it is difficult to determine the reliability and validity of the original information. Indirect evidence for the adequacy of the data is provided by expected relations among medical measures, but it is important to replicate these results with more consistent measurement. It may be easier to document relations with indicators of prenatal than postnatal androgen excess because the former are easier to measure than the latter, especially from retrospective reports. Despite these limitations, our results are consistent with expectations and with other studies in females with CAH using less extensive assessments regarding associations between behavior and disease characteristics (6, 17, 38).
We are continuing to test in other ways the relative importance for different behaviors of prenatal vs. postnatal androgens. This includes studies of girls with CAH varying in treatment: girls who received prenatal glucocorticoid treatment and thus have low prenatal androgen levels (32, 44) and girls who received adrenalectomy and thus have low postnatal androgen levels (45). We are also following girls detected through newborn screening who represent a population sample and for whom medical information can be obtained prospectively rather than retrospectively (46).
In conclusion, our results are most consistent with the hypothesis that behavioral masculinization in females with CAH, especially with respect to toy play, results from high levels of androgens in fetal development and not in postnatal life. They confirm results in other mammalian species that the sensitive periods for androgen effects on behavior and the brain occur early in development (19, 20, 21).
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Acknowledgments |
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Footnotes |
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Received March 9, 1999.
Revised October 7, 1999.
Accepted November 8, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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