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Sex Begins in the Womb ABSTRACT Sex differences of importance to health and human disease occur through- out the life span, although the specific expression of these differences varies at different stages of life. Some differences originate in events occurring in the intrauterine environment, where developmental processes differentially organize tissues for later activation in the male or female. In the prenatal period, sex determination and differentiation occur in a series of sequential processes governed by genetic and environmental factors. During the pu- bertal period, behavioral and hormonal changes manifest the secondary sexual characteristics that reinforce the sexual identity of the individual through adolescence and into adulthood. Hormonal events occurring in puberty lay a framework for biological differences that persist through life and that contribute to variable onset and progression of disease in males and females. It is important to study sex differences at all stages of the life cycle, relying on animal models of disease and including sex as a variable in basic and clinical research designs. All human individuals whether they have an XX, an XY, or an atypi- cal sex chromosome combination begin development from the same starting point. During early development the gonads of the fetus remain undifferentiated; that is, all fetal genitalia are the same and are phenotypi- cally female. After approximately 6 to 7 weeks of gestation, however, the expression of a gene on the Y chromosome induces changes that result in the development of the testes. Thus, this gene is singularly important in 45

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46 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH inducing testis development. The production of testosterone at about 9 weeks of gestation results in the development of the reproductive tract and the masculinization (the normal development of male sex character- istics) of the brain and genitalia. In contrast to the role of the fetal testis in differentiation of a male genital tract and external genitalia in utero, fetal ovarian secretions are not required for female sex differentiation. As these details point out, the basic differences between the sexes begin in the womb, and this chapter examines how sex differences develop and change across the lifetime. The committee examined both normal and abnormal routes of development that lead individuals to become males and females and the changes during childhood, reproductive adulthood, and the later stages of life. BIOLOGY OF SEX One of the basic goals of biologists is to explain observed variability among and within species. Why does one individual become infected when exposed to a microbiological agent when another individual does not? Why does one individual experience pain more acutely than an- other? Sex is a prime variable to which such differences can be ascribed. No one factor is responsible for variability, but rather, a blend of genetic, hormonal, and experiential factors operating at different times during development result in the phenotype called a human being. As suggested by the reproductive processes of some species and punc- tuated by recent successful efforts at cloning of some species, sexual re- production is not necessary for species perpetuation. Debate exists on why sexual reproduction has evolved. Most biologists agree that it in- creases the variability upon which evolutionary selection can operate; for example, variability would allow some offspring to escape pathogens and survive to reproduce. This theory is not without its critics (Barton and Charlesworth, 1998~. The contribution of genetics to sex differences has been described in Chapter 2. Here the focus is more on the endocrine and experiential bases for the development and expression of sex as a phenotype. Different species of vertebrate animals have evolved different path- ways to determine sex, but it is interesting that in all cases two sexes emerge with distinctly different roles in the social and reproductive lives of the animals (Crews, 1993; Francis, 1992~. In all vertebrates the genetic basis of sex is determined by meiosis, a process by which paired chromo- somes are separated, resulting in the formation of an egg or sperm, which are then joined at fertilization. Variations in the phenotypic characteris- tics of the different sexes are determined during development by internal chemical signals. The process can be influenced by external factors such

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SEX BEGINS IN THE WOMB 47 as maternal endocrine dysfunction or endocrine disrupters, as well as fetal endocrine disorders and exogenous medications (Grumbach and Conte, 1998~. Nongenomic Sexual Differentiation and Sexual Flexibility Nongenomic sexual differentiation has evolved in several species of fishes and reptiles. In these species, sex results from external signals. For example, temperature during embryogenesis is the cue acting on autoso- mal genes to result in adult males and females in several species. In many species of flounder, for instance, elevated temperatures of the water in which the larval fish develop results in a higher proportion of males (Yamamoto, 1999~. Similarly, in several turtle species the incubation tem- perature of the eggs influences the sex ratio of the animals (Crews et al., 1989). In some species, sex determination can be delayed until well after birth or the sex can even change after the birth of an organism. One fascinating study found that several species of fish develop sexual pheno- types as a result of the fish's social rank in a group (Baroiller et al., 1999; Warner, 1984~. The blue-headed wrasse is a polygynous coral reef fish with three phenotypes that vary in size, coloration, reproductive organs, physiology, and behavior (Godwin et al., 1996; Warner and Swearer, 1991~. These phenotypes are females, initial-phase males, and terminal-phase males. As a result of changes in the social role, a fish can progress rapidly through these phenotypes. Upon the disappearance of a terminal-phase male, the behavior of the largest female in the group converts to male-like behavior in minutes and the fish shows full gonadal changes in days. The belted sandfish (Serranus subligarius) stands out as one of the most remarkable demonstrations of vertebrate sexual flexibility. This coastal marine fish is a simultaneous hermaphrodite (Cheek et al., 2000~. Its gonads produce both sperm and eggs, and each fish has the reproduc- tive tract anatomies of both sexes simultaneously. Within minutes each individual can show three alternative mating behaviors that is, female, courting male, or streaker male along with the appropriate external color changes (Cheek et al., 2000~. A streaker male awaits the peak moment during the courtship of male and female morphs and then streaks in to release sperm at the moment of spawning. The sperm compete with the courting male's sperm. Partners can switch between male and female roles within seconds and may take turns fertilizing each other's eggs. The frequency with which an individual plays the female or male role is, in part, a function of size. Larger fish are more likely to play the male role more often. In contrast, mammalian sex determination is more directly under the

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48 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH control of a single internal event: fertilization. Under normal conditions, the direction of sexual development is initiated and determined by the presence or absence of a Y chromosome. Intrauterine Environment In mammals, once genetic sex has been determined and the fetus begins its development, the fetal environment, especially hormones, can result in significant modifications of the genetically based sex. The effect of prenatal hormones on later anatomy, physiology, and behavior are most clearly demonstrated in several animals showing the "intrauterine position effect" (vom Saal et al., 1999~. In litter-bearing mammals such as mice, rats, gerbils, and pigs, each pup shares the uterus with several others, some of which are of a different sex. Significant differences among females occur if the fetus is located between two males or with a male on one side or with no male on either side. Testosterone is produced by fetal males and can masculinize adjacent females to various degrees. Thus, not only do individuals vary as a result of genetic variability, but they can also vary as a result of prenatal hormonal organizational effects (see addi- tional discussion in Chapter 4~. Extensive studies with the female mouse have revealed that adult anatomical structures, such as the genitalia and sexually dimorphic parts of the brain, and the rate of reproductive devel- opment vary as a result of proximity to males in the womb (Vandenbergh and Huggett, 1995~. Studies with animals suggest that hormonal transfer between fetuses can influence later anatomical, physiological, and behavioral characteris- tics. Some data from studies with humans, recently summarized by Miller (1998), suggest that a similar phenomenon occurs in mixed-sex twins. His review of the literature reveals a number of characteristics apparently influenced by transmission of testosterone from the male twin to the fe- male twin. For example, (1) dental asymmetry is also a characteristic of females with male co-twins (the right jaw of the male has larger teeth) (Boklage, 1985), (2) spontaneous otoacoustic emissions are at an interme- diate level in females with male co-twins (the rates of clicking sounds produced in the cochlea usually differ between males and females) (McFadden, 1993), and (3) the level of sensation seeking appears to be higher in females with male co-twins than in those without male co-twins (Resnick et al., 1993~. These studies suggest that, as in rodent models, testosterone transferred to human female fetuses can have masculinizing effects on anatomical, physiological, and behavioral traits. In humans, the metabolic stress of pregnancy increases the incidence of gestational diabetes in susceptible women. Transgenerational passage of diabetes may contribute to the higher incidence of impaired glucose

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SEX BEGINS IN THE WOMB 49 tolerance, obesity, and hypertension in the offspring of diabetic mothers and to the prevalence of diabetes in such human communities as the Pima Indians (Cho et al., 2000; Silverman et al., 1995~. This passage of a disease condition across generations by non-genome-dependent mechanisms emphasizes the importance of good maternal care and health during preg- nancy. Although males will also be affected by a hyperglycemic environ- ment during fetal life and will themselves have an increased risk of diabe- tes in adulthood, they do not provide the womb environment during the critical phases of fetal development of the next generation. Thus, males do not pass the tendency across generations (Cho et al., 2000; Nathanielsz, 1999; Silverman et al., 1995~. Low birth weight or small body size at birth as a result of reduced intrauterine growth are associated with increased rates of coronary heart disease and non-insulin-dependent diabetes in adult life (reviewed by Barker [2000~. The "fetal origins hypothesis" proposes that undernutri- tion during critical periods of fetal growth can force the fetus to adapt by altering cardiovascular, metabolic, or endocrine functions to survive. (Note that debate continues as to whether the association is truly causal [Kramer, 2000; The Lancet, 2001; Lumey, 2001~.) These changes, such as redistribution of blood flow, changes in the production of fetal and pla- cental hormones involved in growth, and metabolic changes, can perma- nently change the function and structure of the body. For example, off- spring who were exposed in utero to maternal famine during the first trimester of development had higher total cholesterol and low-density lipid cholesterol levels and a higher ratio of low-density lipid to high- density lipid cholesterol levels, all of which are risk factors for heart dis- ease (Roseboom et al., 2000~. This altered lipid profile persisted even after adjustments for adult lifestyle factors such as smoking, socioeconomic status, or use of lipid-lowering drugs. Male offspring had higher rates of obesity at age 19 years, but maternal malnutrition during early gestation was associated with a higher prevalence of obesity in 50-year-old women (Ravelli et al., 1999~. Such permanent alterations in body structure or functions may have effects on future generations as well. Studies show that when a female fetus is undernourished and subsequently of low birth weight, the perma- nent physiological and metabolic changes in her body can lead to reduced fetal growth and raised blood pressure in her offspring (Barker at al., 2000; Stein and Lumey, 2000~. Furthermore, in birth cohorts of males with spine bifida who had been exposed to prenatal famine, the relative risk of death was 2.5-fold greater than that in similarly affected female offspring (Brown and Susser, 1997~. These traits in the offspring were not affected by the father's size at birth.

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50 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH EARLY DEVELOPMENT The remarkable accumulation of knowledge over the past five de- cades and new and continuing insights in the field of sex determination and sex differentiation represent major landmarks in biomedical science. No aspect of prenatal development is better understood. Advances in embryology, steroid biochemistry, molecular and cell biology, cytogenet- ics, genetics, endocrinology, immunology, transplantation biology, and the behavioral sciences have contributed to the understanding of sexual anomalies in humans and to the improved clinical management of indi- viduals with these disorders. Major contributions to this understanding have stemmed from studies of patients with abnormalities of sex determi- nation and differentiation and the recent advances emanating from mo- lecular genetics. These advances, considered together, illustrate that a failure in any of the sequential stages of sexual development, whether the cause is genetic or environmental, can have a profound effect on the sex phenotype of the individual and can lead to complete sex reversal, vari- ous degrees of ambisexual development, or less overt abnormalities in sexual function that first become apparent after sexual maturity (Grumbach and Conte, 1998; Wilson, 1999~. Sex Determination Sex determination and sex differentiation are sequential processes that involve successive establishment of chromosomal sex in the zygote at the moment of conception, determination of gonadal (primary) sex by the genetic sex, and determination of phenotypic sex by the gonads. At pu- berty the development of secondary sexual characteristics reinforces and provides more visible phenotypic manifestations of the sexual dimor- phism. Sex determination is concerned with the regulation of the develop- ment of the primary or gonadal sex, and sex differentiation encompasses the events subsequent to gonadal organogenesis. These processes are regulated by at least 70 different genes that are located on the sex chromo- somes and autosomes and that act through a variety of mechanisms in- cluding those that involve organizing factors, gonadal steroids and pep- tide hormones, and tissue receptors. Mammalian embryos remain sexually undifferentiated until the time of sex determination. An important point is that early embryos of both sexes possess indif- ferent common primordia that have an inherent tendency to feminize unless there is active interference by masculinizing factors (Grumbach and Conte, 1998~. It has been known for more than four decades that a testis-determin- ing locus, TDF (testis-determining factor), resides on the Y chromosome. About 10 years ago, the testis-determining gene was found to be the SPY

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SEX BEGINS IN THE WOMB 51 (sex-determining region Y) gene (Ferguson-Smith and Goodfellow, 1995; Koopman, 1999; Koopman et al., 1991; O'Neill and O'Neill, 1999; Sinclair et al., 1990; Swain and Lovell-Badge, 1999), which is the primary sex determinant, as it is the inducer of differentiation of the indifferent gonad into testes and hence is the inducer of male sexual development. SRY is expressed in 46,XY gonads in Sertoli cell progenitors at the stage of sex cord formation, but unlike the mouse, in which SRY expression is brief, SRY mRNA persists in Sertoli cells at 18 weeks of gestation (Hanley et al., 2000~. As discussed in Chapter 2, the human SRY gene is located on the short arm of the Y chromosome and comprises a single exon that encodes a protein of 204 amino acids including a 79-residue conserved DNA bend- ing and DNA binding domain: the HMG (high-mobility-group) box. The mechanisms involved in the translation of genetic sex into the development of a testis or an ovary are now understood in broad terms (Figure 3-1~. It is known that a variety of autosomal and X-chromosome-linked genes, literally a cascade of genes that exert complex gene dosage balanc- ing activities, are involved in testis determination. All major sex-deter- mining genes have been shown to be subject to a dosage effect. In the human, the SRY protein is detected at an early age of gonadal differentia- tion in XY embryos, where it induces Sertoli cell differentiation. In the human adult, it is present in both Sertoli and germ cells. In embryonic and fetal life, the evidence suggests that the SRY gene product regulates gene expression in a cell-autonomous manner. The precise molecular mecha- nisms by which SRY triggers testis development are unknown, nor is it yet known how SRY is regulated. The genetic sex of the zygote is estab- lished by fertilization of a normal ovum by an X-chromosome- or Y- chromosome-bearing sperm. Genes Contributing to Sex Determination Apart from SRY, a number of autosomal and X-chromosome-linked genes have been identified and have a critical role in male or female sex determination, the testis- and ovary-determining cascades (Roberts et al., 1999) (Table 3-1~. In the human, heterozygous mutations or deletion of the Wilm's tumor (WT1) gene located on chromosome llpl3 results in uro- genital malformations as well as Wilm's tumors. Knockout of the WT1 gene in mice results in apoptosis of the metanephric blastema, with the resultant absence of the kidneys and gonads. Thus, WT1, a transcriptional regulator, appears to act on metanephric blastema early in urogenital development. SF-1 (steroidogenic factor-1) is an orphan nuclear receptor involved in transcriptional regulation. It is expressed in both the male and the female urogenital ridges as well as steroidogenic tissues, where it is re-

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52 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH Permission was not granted to electronically reproduce figure 3-1 from /n:Wi//iams Textbook of En docrino/ogy, 9th eel. J.D. Wilson, D.W. Foster, H.M. Kronenberg, and P.R. Larsen, eds. Philadelphia: W.B. saunclers Co. This figure is available in the printed version of this report.

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53 CD o o o . - x cry o H V) cry Em CD ~ O ~ .tn .`n CD 5-d ~ CD CD ~ ~ ~ ~ 3 ~ x ~ X 5- Co Co so ~ X. .`n ~ ~ O ~ ~ m ~ m ~ E ~ ~ ~ C ~ ~ ~ ~ o so ,' ~ ad, ~ o ~ :^ X ~ ~ ~ ~ X ~ o . - u 5- 5- 5- 5- 5- 5-1 ~ o o o o o o , U U U U U U ~ o o o o o o o ,~ u u u u u u u co co co cn cn cn 5- 5- 5- 5- 5- 5- x 5- 5- o o ~ ~ - -d u u ~ . ~ ~ ~ o o ~ ~ 5 o ,, a C c ~ E ~o o . o o o ~ ~ ~ X - - C ~ ~ ~ o ~ ~

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54 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH quired for the synthesis of, for example, testosterone and estrogen, and in Sertoli cells, where it regulates the anti-mullerian hormone gene (Parker et al., 1999~. SF-1 is encoded by the mammalian homologue of the Dro- sophila melanogaster gene (FTZ-F1~. Knockout of the Sf-1 gene in mice re- sults in apoptosis of the genital ridge cells that give rise to the adrenals and gonads and, thus, a lack of gonadal and adrenal morphogenesis in both males and females. A heterozygous mutation in the human gene encoding SF-1 causes XY sex reversal (Achermann et al., 1999), which results in individuals with normal female external genitalia, streak-like gonads containing sparse and poorly differentiated tubules, and the fail- ure of adrenal development. WT1 and SF-1 appear to play important roles in the differentiation of the genital ridge from the intermediate meso- derm. WT1 and SF-1 are expressed when the indifferent gonadal ridge first differentiates at 32 days postovulation in both female and male em- bryos (Hanley et al., 1999~. XY gonadal dysgenesis with resulting female differentiation has oc- curred in 46,XY individuals with intact SRY function but with duplication of Xp21, leading to a double dose of the DAX-1 (dosage-sensitive sex reversal congenital adrenal hypoplasia congenital-critical region on the X chromosome, gene 1) gene. On the other hand, a mutation or deletion of DAX-1 in XY individuals results in X-linked congenital adrenal hypopla- sia and hypogonadotropic hypogonadism but not an abnormality in testis differentiation. Similarly, duplication of the DAX-1 gene on one X chro- mosome appears not to affect ovarian morphogenesis or function in 46,XX females. Targeted disruption of the Dax-1 gene in mice does not affect ovarian development. It has been suggested that Dax-1 is an "anti-testis" factor rather than an ovary-determining gene. SRY and Dax-1 appear to act antagonistically in gonadal dysgenesis (Parker et al., 1999; Roberts et al., 1999~. Dax-1 expression is detected in the primate gonadal ridge days before the peak expression of SRY (Hanley et al., 2000~. Camptomelic dysplasia is a skeletal dysplasia associated with sex reversal because of gonadal dysgenesis in about 60 percent of affected 46,XY individuals. A gene for a camptomelic dysplasia, SOX-9, has been localized to 17q24.3-25.1. The products of SOX genes (for the SRY-related HMG-box gene), as a rule, are more than 50 percent identical to those of SRY genes at the amino acid level in the HMG-box region (Koopman, 1999~. In the human, SOX-9 transcripts are present in the gonadal ridge of both male and female embryos (Hanley et al., 2000~. XY individuals with 9p- or 10q- deletions as well as patients with lp32-36 duplications exhibit gonadal dysgenesis and male pseudoherma- phrodism, which suggests that autosomal genes at these loci are impor- tant in the gonadal differentiation cascade. In this regard, two genes, DMRT-1 and DMRT-2, have been localized to the distal region of the short arm of chromosome 9 (Raymond et al., 1999~. These genes are re-

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SEX BEGINS IN THE WOMB 55 fated to the sexual regulatory genes Dsx (double sex) in D. melanogaster and MAB-3 in Caenorhabditis elegans (or double sex- and MAB-3-related transcription factor). Their evolutionary conservation, deletion from sex- reversed males with the 9p- syndrome, and male-specific expression in early human gonadogenesis suggest that one or both genes have a role in human sex determination (Calvari et al., 2000~. WNT-4, a vertebrate homologue of the D. melanogaster polarity gene ("wingless"), is involved in the regulation of steroid biosynthesis in the fetal gonad (Uusitalo et al., 1999~. Wnt-4 knockout female mice lack mullerian ducts and exhibit decreased levels of oocyte development and decreased rates of survival. WNT-4 is downregulated in the fetal testis, presumably by SRY. Consequently, testosterone synthesis occurs in the XY individual. It has recently been demonstrated that WNT-4 in humans is located on chromosome lp35 and that duplication of WNT-4 upregu- lates DAX-1 expression and causes sex reversal in a 46,XY individual. 46,XX mice with homozygous disruption of the Wnt-4 gene manifest tes- tosterone synthesis in the fetal ovary and masculinization of the wolffian ducts. This observation suggests that Wnt-4 expression in the fetal ovary inhibits gonadal androgen biosynthesis. Organogenesis of the Testes Until about the 12-millimeter stage (approximately 42 days of gesta- tion), the embryonic gonads of males and females are indistinguishable. By 42 days, 300 to 1,300 primordial germ cells have reached the undiffer- entiated gonad from their extragonadal origin in the dorsal endoderm of the yolk sac. These large cells are the progenitors of oogonia and sper- matogonia. In the absence of primordial germ cells, the gonadal ridges in the female remain undeveloped. Germ cells are not essential for differen- tiation of the testes (Grumbach and Conte, 1998~. There is a striking sexual dimorphism in the timing of gonadal differ- entiation under the influence of SRY and other testis-determining genes (Figures 3-2 and 3-3~. Organization of the indifferent gonad is definitive by the 6th to 7th week of gestation; the testes develop more rapidly than the ovaries. The ovary does not emerge from the indifferent stage until 3 months of gestation, when the earliest sign of differentiation into ovaries appears: the beginning of meiosis, as evidenced by the maturation of oogonia into oocytes. The precursor of the Sertoli cell that arises from the coelomic epithelium expresses SRY, leading to differentiation of Sertoli cells, which marks testis differentiation (Caper, 2000~. The Sertoli cell is the only cell in the testes in which SRY has a critical effect. Germ cells in the XY gonad are sequestrated inside the forming testis cords. Anti- mullerian hormone (AMH) (or mullerian-inhibiting factor or substance) is a member of the transforming growth factor beta family, one of the

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68 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH growth spurt in boys as well as girls (reviewed in Grumbach [2000] and Grumbach and Auchus [1999~. In boys, the estradiol is derived mainly from the extragonadal conver- sion of testosterone to estradiol in a wide variety of tissues, but there is also a small testicular contribution (Siiteri and MacDonald, 1973~. Fur- thermore, estradiol, but not testosterone, appears to be the critical media- tor of skeletal maturation and epiphyseal fusion and the major sex steroid in bone mineral accrual in boys as well as girls (Grumbach, 2000; and Grumbach and Auchus, 1999~. This conceptual sea change has emanated from studies of men, women, and children with mutations in the gene encoding aromatase (Bilezikian et al., 1998; Grumbach and Auchus, 1999; Morishima et al., 1995) and from studies of one man with a null mutation in the gene encoding the estrogen receptor or (Smith et al., 1994~. There is a very striking and poorly understood difference in the preva- lence of so-called idiopathic true or central precocious puberty in boys and girls. The idiopathic form is about 10 times more common in girls than in boys. In contrast to the striking sex difference in idiopathic true precocious puberty, constitutional delay in growth in adolescents (idio- pathic delayed puberty) is more common in boys than in girls. Adrenarche Versus Gonadarche In both boys and girls, beginning before age 8 (skeletal age, 6 to 8 years), an increase in the levels of secretion of adrenal androgens and androgen precursors, called "adrenarche," occurs. It is marked biochemi- cally by progressive increases in plasma dehydroepiandrosterone and

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SEX BEGINS IN THE WOMB 69 Permission was not granted to electronically reproduce figure 3-5 from In:Williams Textbook of Endocrinology, 9th ed. J.D. Wilson, D.W. Foster, H.M. Kronenberg, and P.R. Larsen, eds. Philadelphia: W.B. launders Co. This figure is available in the printed version of this report. dehydroepiandrosterone sulfate (DHEAS) concentrations. The mecha- nism of activation of adrenal androgen secretion or adrenarche is inde- pendent of the mechanisms that regulate the onset of sex steroid secretion by the gonads, which is called "gonadarche" (Grumbach and Styne, 1998~. Premature adrenarche, which is more common in girls than in boys, is characterized by the precocious appearance of pubic hair or axillary hair, less commonly an apocrine odor, and comedones and acne without other signs of puberty or virilization (Grumbach and Styne, 1998~. Adre- narche is premature when it occurs in Caucasian girls before age 7 or African-American girls before age 5. In boys the diagnosis is limited to those who develop pubic hair or axillary hair before the age of 9. In contrast to boys, in whom premature adrenarche is usually a benign, self-

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70 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH limited normal variant of puberty, girls with premature adrenarche are at increased risk (about 10-fold) for the development of insulin resistance and ovarian hyperandrogenism, in particular, polycystic ovary syndrome (PCOS) (Dunaif et al., 1992; Ibanez et al., 1996; Morales et al., 1996; Oppenheimer et al., 1995~. PCOS affects about 5 to 10 percent of women of reproductive age and is the most common endocrine disorder in women. A proportion of girls with exaggerated adrenarche (Likitmaskul et al., 1995), which is marked by higher levels of circulating DHEAS, may ex- hibit insulin resistance and hyperinsulinism or dyslipidemia, and begin- ning in late adolescence they are at increased risk for the development of ovarian hyperandrogenism and anovulation (Ibanez et al., 1998, 1999a). Affected girls, however, usually begin gonadarche within the normal range of time. There is a correlation between the occurrence of exaggerated adren- arche in prepubertal girls and a higher risk for ovarian hyperandrogenism at puberty. The androgen excess is a consequence of PCOS and is associ- ated with an increased risk of metabolic complications, including type II diabetes mellitus, hypertension, dyslipidemia, and possibly, cardiovascu- lar disease. There is a tendency for familial aggregation of women with PCOS, and evidence suggests that this heterogeneous disorder represents a complex, multifactorial trait. A promising area of research is the increasing body of evidence that supports an association of premature adrenarche, insulin resistance, and dyslipidemia in girls with intrauterine growth retardation (Francois and de Zegher, 1997; Ibanez et al, l999b). Recent studies (Ibanez et al., 2000) suggest that girls with prenatal growth restriction have at birth a smaller complement of primordial follicles than infants of appropriate weight for gestational age and have at adolescence a small uterus and ovaries. After menarche these girls tend to show ovarian hyporesponsiveness to FSH. In sum, the evidence suggests a link between intrauterine growth retardation and the increased risk of exaggerated adrenarche followed by PCOS, including hyperandrogenism, insulin resistance, dyslipidemia (with or without obesity), and cardiovascular disease (Barker, 1995, 1997; Cresswell et al., 1997~. As first advanced by Barker (1995) from observa- tional studies, the association of impaired or disproportionate fetal growth, related to fetal undernutrition, with premature adrenarche and PCOS is another example of disorders in adolescence and adulthood that may be programmed in fetal life. Many issues, however, remain unre- solved (laquet et al., 1999~. Sex Differences in Behavior The hormonal and physical changes at puberty described above have implications for sex differences in behavior in early adolescence. Some

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SEX BEGINS IN THE WOMB 71 behavioral changes probably result from the direct effects of gonadal hor- mones acting directly on the brain. For example, in early adolescence, increasing testosterone levels in boys have been associated with increas- ing aggression and social dominance, and changes in estrogen levels in girls have been associated with mood changes (Brooks-Gunn et al., 1994; Buchanan et al., 1992; Finkelstein et al., 1997; Olweus et al., 1980; Schaal et al., 1996; Susman et al., 1987~. Some behaviors appear to relate to the absolute level of the hormone, others appear to relate to the ratio of hor- mone levels (for example, the testosterone level/estradiol level ratio), and others appear to relate to hormonal variation. (These associations are not always noted and probably depend on the reliability of hormone level measurements, intersubject variation, and the specific behaviors mea- sured.) It is important to note that hormone levels themselves can be changed by behavior. For example, winning an athletic event has been shown to increase testosterone levels in males (Booth et al., 1989~. As discussed later, the rise in estrogen levels at puberty may contrib- ute to females' superior phonological skills and may allow females with dyslexia to compensate for their reading deficiencies. There is also some suggestion that other cognitive changes in adolescence are related to hor- monally induced maturation of the frontal lobe (Spear, 2000~. Given the sex difference in the timing of gonadarche, there may well be sex differ- ences in the developmental timing of behaviors subserved by the frontal lobe (including planning and judgment), although probably not in the ultimate levels of those behaviors at maturity. The hormonal and physical changes that occur during puberty also contribute in indirect ways to differences between adolescent boys and adolescent girls. For example, the development of secondary sex charac- teristics in girls creates social signals that result in different responses from peers, parents, and teachers. There is a substantial literature show- ing that girls who mature earlier than their peers are at greater risk than girls who mature on time or later than their peers for problems during the pubertal transition and continuing into adulthood. These problems in- clude substance use, depression, and eating disorders (Caspi and Moffitt, 1991; Graber et al., 1997~. Some of these effects are mediated by the fact that girls who mature early are more likely than others to associate with older adolescents and to be treated as if they are older (including in- creased responsibilities from parents and increased expectations of par- ents). Boys who mature earlier than their male peers do not have a simi- larly increased risk of problems compared with the risk for boys who mature on time or later, in part because the absolute age of boys who mature early is, on average, 2 years later than that of girls who mature early and because their physical maturation gives them status among adolescents, who value athleticism and physical skill in boys. Adolescence is associated with changing social roles, and there is

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72 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH good reason to believe that gender socialization intensifies at that time of life (Crouter et al., 1995; Hill and Lynch, 1983; Stein, 1976~. These changes in social roles will have wide-ranging effects, including, for example, variations in interpretations of harmful stimuli and responses to injury, as discussed below. ADULTHOOD During the long period of about 40 years of fertile adulthood, an individual's occupations, social roles, and lifestyle change episodically and develop slowly as experiences accumulate. Although societal norms are rapidly changing, in general it remains the case that women still pre- dominate as caregivers and organizers with wide-ranging obligations and duties spanning the family, workplace, and leisure realms, whereas men still predominate in more focused aggressive and physically demanding activities with a relatively narrower range of social obligations. Accompa- nying these developing and highly individual psychosocial characteris- tics are the more consistent (but not constant) sex differences in anatomy, organ function (physiology), and endocrine function. Thus, on average, women, relative to men, have a higher percentage of body fat, smaller muscle mass, lower blood pressure, higher levels of estrogens and pro- gestins, and lower levels of androgen. The challenge in understanding the significance of this vast array of sex differences for health and health care lies not so much in assessing the influence of each of these factors in isolation but, rather, in deciphering how the factors interact throughout the course of adulthood to affect each individual at any moment. In addition, women, but not men, undergo fluctuations associated with the reproductive condition (such as the ovarian cycle and pregnancy) that influence numerous bodily functions (e.g., gastrointestinal transit time, urinary creatinine clearance, liver enzyme function, and thermo- regulation), including brain function. (The effects of pregnancy, lactation, and parity are obviously important to the health of women later in their lives but are not addressed specifically in this report.) Effects of Menopause on Women's Health After the fertile years in women there is a 5- to 10-year period of menopause-related alterations in hormone patterns, terminating in the sharp decline in female hormone levels. As follicle depletion occurs in the ovaries, the rate of ovarian hormone production slows. The tissues most affected by reduced estrogen levels are the ovaries, uterus, vagina, breast, and urinary tract. However, other tissues such as the hypothalamus, skin, cardiovascular tissue, and bone are also substantially affected. A major

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SEX BEGINS IN THE WOMB 73 challenge to the prevention of disease in older women lies in exploring the effects of both short-term and long-term reductions in ovarian hor- mone levels on the development of symptoms and disease. The lack of ovarian estrogens appears to contribute significantly to the onset of several postmenopausal diseases, such as osteoporosis and cardiovascular disease, two leading causes of morbidity and mortality in older women. Much of the evidence to support the finding of a cardio- protective effect for estrogen has come from observational studies of women on estrogen replacement therapy, which has shown that estrogen users experience half as many cardiovascular events as nonusers, but numerous questions remain (also discussed in Chapter 5~. An adverse influence of hormone therapy on cardiovascular risk in women with coro- nary heart disease has been shown during the initial year of use; however, few data are available on the effects of long-term hormone therapy (Grodstein et al., 2000~. The protection conferred by estrogen has been shown to be mediated by mechanisms acting at different levels, including a beneficial effect of estrogen on plasma lipid concentrations (Lamon- Fava, 2000~. In addition, research has identified estrogen receptors in bone (re- viewed in Grumbach and Auchus, 1999; Khosla et al., 1999; Prestwood et al., 2000~. Declines in estrogen production correlate with rapid bone loss, which predisposes a woman to osteoporosis. Although age-related bone loss is a universal phenomenon shared by men and women, the effect of osteoporosis on women is much more profound and pervasive. Several reports have shown that combining high-calcium supplements with a regimen of hormone therapy increases the efficacy of estrogen in bone conservation. Hormone replacement by estrogen therapy or the newly developed therapy with selective estrogen receptor modulators may pre- vent the development of osteoporosis and its related fractures (Kamel et al., 2001~. In men, more inconsistent and complex changes in hormone metabo- lism, called "andropause" by some, occur over a longer period of time, on average, between the ages of 48 and 70 (Morales et al., 2000; Vermeulen, 2000~. Currently, much more is known about the consequences of meno- pause than about those of andropause. Androgen deficiency has been shown to be associated with osteoporosis. Although testosterone replace- ment therapy in hypogonadal men decreases bone resorption and in- creases bone mass, placebo-controlled trials are needed to better define the effectiveness and risks of such therapy in older men. The effect of testosterone is, at least in part, related to its conversion in bone (Bilezikian et al., 1998; Grumbach, 2000; Khosla et al., 1998; Smith et al., 1994) to estradiol.

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74 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH The Aging Human The evolving effects of interactions between an individual's personal physiology and unique experiences make it difficult to assess how simply being female or male affects that individual's health as life progresses from birth through fertile adulthood into old age. Although the field of gerontology is growing rapidly, research from this perspective is meager. Most studies simply address the question of how elderly individuals differ from younger individuals, with little attention paid to how the differences might develop over time. Nevertheless, it is evident that the patterns of sex differences that exist during the long period of fertile adulthood change during old age in clinically relevant ways. For example, community prevalence estimates for chronic widespread pain and fibro- myalgia show a general increase with age until about age 65, followed by a decrease, with the prevalence in women always being higher (LeResche, 1999~. On the other hand, the prevalence of pain in the knee or finger joints shows a continual increase across the life span for both sexes, with no sex differences until age 50, after which the prevalence becomes higher in women (LeResche, 1999~. A second example in this case, one relevant to diagnosis is that the symptom presentation of patients with confirmed acute myocardial infarction varies by sex, but, importantly, the pattern changes with age (Goldberg et al., 2000~. Younger patients (less than age 55) were significantly more likely than older patients to complain of sweat- . , . ng ana arm Pam. A third example in this case, relevant to treatment involves recent data showing sex- and age-related differences in the optimal effects of antihypertensive and antiplatelet therapies for the prevention of cardio- vascular disease (Kjeldsen et al., 2000~. For example, compared with treat- ment with a placebo, daily acetylsalicylic acid (ASA) treatment resulted in a significant reduction in the rate of occurrence of composite major car- diovascular events in younger patients (younger than age 65~. Some re- duction in the rate of occurrence of major cardiovascular events was also seen in ASA-treated older patients, but the reduction was not statistically significant. From 1900 through about 1940, Americans who lived to age 65 had a life expectancy of another 11 or 12 years, regardless of sex. Since the 1940s differences in life expectancies between males and females after age 65 have emerged, and these differences favor females. Similarly, in 1900 individuals who reached the age of 85 had, on average, another 4 years of life, with very little difference between the sexes. Differences in survival began to appear in the 1960s, and these again favored females. Much of this difference can be attributed to differences in rates of death from car- diovascular disease. A breakdown by sex and age (65 to 74 years, 75 to 84 years, and 85 years and older) reveals that in each age group men have

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SEX BEGINS IN THE WOMB 75 higher death rates from both heart disease and cancer than women (Na- tional Center for Health Statistics, 1999~. Death rates from stroke, another leading cause of death, are more balanced between males and females. Currently, life expectancy at birth is greater for females than males by ~6 years, but once old age has been attained, it becomes 2 to 3 years (Table 3-3~. The actual life expectancy differs among ethnic groups and is, for example, notably shorter among African American than Caucasian Ameri- cans, but the consistency in the observation of an advantage for females across ethnic groups is striking (Figure 3-6~. This consistent observation of greater life expectancy at birth for females has grown over time, from about 2 years in 1900 to 6 years in 1998 (Figure 3-7), with some fluctua- tions during the interim. Although the mechanisms that underlie both the general increase in longevity and the increasing advantage for females are poorly under- stood, some components of the male longevity disadvantage can be iden- tified. For example, rates of death from the major causes (both intentional and unintentional injuries and illnesses) are usually higher for males than for females at each stage of life (reveille et al., 2000), although there are exceptions in which the rates are similar for the two sexes or are higher for females. Stress and its hormonal consequences are complex factors that may contribute to longevity. A recent provocative suggestion is that the be- havioral response to stress may differ between males and females (Taylor et al., 2000~. According to this hypothesis males display the classic "fight- or-flight" response that in females is modulated to become a "tend-and- defend" response. On the basis of a meta-analysis (integrating the data from a number of independent studies), the female's response is appar- ently mediated by oxytocin, a hormone known to reduce stress and in- crease social affiliation in rodents (Carter et al., 1995; Will et al., 1990~. This proposed difference in response may have implications for the sex TABLE 3-3 Life Expectancy at Birth, Age 65, and Age 75 Years, United States, All Races, 1998 Life Expectancy (years) Difference (Female Percent Age Males Females minus Male) Differencea At birth 73.8 79.5 5.7 7 At age 65 16.0 19.2 3.2 17 At age 75 10.0 12.2 2.2 18 a Percent difference equals (life expectancy for females minus life expectancy for males) divided by life expectancy for females. SOURCE: National Center for Health Statistics (2000a).

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76 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH Am Indian/Alaska Native Native Hawaiian Samoan Guamanian Hispanic Origin Puerto Rican Black U.S. Virgin Islands Black Japanese R Males ~ Females , TO 7 50 55 60 65 70 75 80 85 90 Life Expectancy at Birth (years) FIGURE 3-6 Life expectancy at birth for males and females in several U.S. eth- nic groups (data are from 1989 to 1994~. Source: National Center for Health Statis- tics (1996) and National Institutes of Health, Office of Research on Women's Health (1998~. 90 1 80 70 60 50 40 30 Males Females 48.3 1 77 4 78.8 79.5 73.1 74.7 71.1 ~ 70.0 ~ 7 '1.8 _ 73.8 1900 1950 1960 1970 1980 1990 1998 Calendar Year FIGURE 3-7 Life expectancy at birth for males and females, selected years be- tween 1900 and 1998, United States, all races. Source: National Center for Health Statistics (2000a).

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SEX BEGINS IN THE WOMB 77 differences in stress-related disorders in human populations and may contribute to the longer life span of females. The female longevity advantage, however, is not without cost. A1- though females live longer, those who do live longer experience more disabling health problems than males. Thus, a recent study of 10,263 older adults in three communities in the United States showed that the propor- tion of disabled women increased from 22 percent at age 70 to 81 percent at age 90, whereas the figures for men were 15 and 57 percent, respec- tively (reveille et al., 2000~. Similarly, another study showed that although the current life expectancy at age 32 is 39.45 years for men and 44.83 years for women, it becomes 31.8 years for men and 33.1 years for women when life expectancy is adjusted for "quality of life." In other words, a 5.38-year advantage for women is reduced to 1.3 years (Kaplan and Erickson, 2000~. Clearly, studies that address the mechanisms that underlie the devel- opment of these differences as individuals age could yield important in- formation of benefit to both sexes. FINDINGS AND RECOMMENDATIONS Findings Sex differences occur throughout the life span, although their specific expression varies at different at stages of life. Intrauterine environment: Some sex differences originate in events that begin in the womb, where developmental processes differentially organize tissues for later activation.. Early development: During the prepubertal period there are be- havioral as well as subtle hormonal sex differences. Puberty: During sexual maturation, hormones activate organ sys- tems differently between males and females; these include brain anatomy and functions that were previously organized by hormones and modified by the environment. Adulthood: Throughout the life span, including midlife and old age, the brain, as well as many other organs, retain plasticity and continue to be modified by gene expression, hormones, and environmental factors. These factors act in an integrative manner but may be expressed differ- ently in males and females. To continue to advance human health and health care, research on sex differences in health and illness across the life span is essential. Such research can be aided by information obtained from the observation and study of other species. In this regard, the committee makes the following recommendations.

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78 EXPLORING THE BIOLOGICAL CONTRIBUTIONS TO HUMAN HEALTH Recommendations RECOMMENDATION 2: Study sex differences from womb to tomb. The committee recommends that researchers and those who fund research focus on the following areas: inclusion of sex as a variable in basic research designs, expansion of studies to reveal the mechanisms of intrauterine effects, and encouragement of studies at different stages of the life span to determine how sex differences influence health, illness, and longevity. Sex is an important marker of individual variability. Some of this sex- related variability results from events that occur in the intrauterine envi- ronment but that do not materialize until later in life. Current research varies in its level of attention to these matters. The committee acknowledges that inclusion of people, animals, or cells and tissues of both sexes in all studies is not feasible or appropriate. Rather, the committee is urging researchers to regard sex, that is, being male or female, as an important basic human variable that should be considered when designing, analyzing, and reporting findings from stud- ies in all areas and at all levels of biomedical research. Statistical methods can be used to evaluate the effect of sex without necessarily doubling the sample size of every study. In addition, it is particularly important that researchers revisit and revise approaches to studying whole-animal physi- ology in light of what has been learned in the past decade about major sex differences. RECOMMENDATION 3: Mine cross-species information. Researchers should choose models that mirror human sex differ- ences and that are appropriate for the human conditions being ad- dressed. Given the interspecies variation, the mechanisms of sex differ- ences in nonhuman primates may be the best mimics for some mechanisms of sex differences in humans. Continued development of appropriate animal models, including those involving nonhuman pri- mates, should be encouraged and supported under existing regulations and guidelines (see the Guide for the Care and Use of Laboratory Ani- mals [National Research Council, 19961~. Researchers should be alert to unexpected phenotypic sex differ- ences resulting from the production of genetically modified animals. Sex differences and their relevance to human health can be examined through the use of cross-species comparisons. The use of appropriate animal models can reveal underlying mechanisms of normal and patho- genic processes.