Assessment of Skeletal and Sexual Maturity: Theoretical and Practical Aspects

Milo Zachmann Department of Pediatrics, University of Zurich, Kinderspital, Ziirich, Switzerland

Abstract. Physical changes of sexual maturation and their clinical relevance are discussed in the first part, and the timetable of development in Swiss girls and boys as established from the Zurich longitudinal study is presented. The second part deals with changes in body size and shape and aspects of skeletal maturation. Prepubertal height is nearly identical in both sexes. During puberty important sex differences become evident and maturation is faster in girls by about 2 years. Puberty bigins at the same stage of skeletal maturation (sesamoid bone) in both sexes, and milestones (e.g. peak height velocity) are reached at the same degree of maturation. For bone age estimation, the Greulich and Pyle method is frequently used, but depending on the situation, that of Tanner et al. may be more useful. For height prediction, the methods of Bayley and Pinneau, Roche et al. and Tanner et al. are most widely used. None of them is always the most accurate one, but depending on the condition and relation between bone and chronologic age, the criteria for selection are discussed. Sex hormones modulate body proportions but have no influence on adult height. Adult height and velocity of maturation are independent multifactorial variables. The sex difference in adult height (12.5cm in Switzerland) is due to several components. Puberty is also characterized by changes in body composition. Lean body mass increases early in boys and girls. In girls, it peaks at menarche and then diminishes. The amount and distribution of water increases in boys and decreases in girls secondary to the greater accumulation of fat in girls. Hormonal changes during puberty are discussed in the last part. Basal gonadotropin levels increase, FSH more in girls than in boys, LH about equally in both sexes. More important is the advent of rhythmic, pulsatile gonadotropin secretion. The most obvious changes are the increasing secretion rates of sex steroids. To avoid extreme variation of normal values, estradiol and testoserone should be related to bone age and/or pubertal stages rather than to chronologic age. The role of DHEA in puberty is unclear. It increases before puberty at adrenarche. Also growth hormone, its binding protein, and IGF I and its binding protein 3 increase. Finally, synergistic metabolic effects between GH and sex hormones with examples using the stable isotope 15N are discussed.

Key words: puberty, growth, bone age, height prediction

Puberty is, apart from the fetal period, the time during human life where the most

Correspondence: Milo Zachmann, M.D. Professor of Pediatric Endocrinology Department of Pediatrics University of Zurich Kinderspital Steinwiesstr. 75 CH-8032 Ztirich Switzerland

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dramatic somatic changes take place. If one only occurs before menarche. It can now be looks at the simplest thing, the weight of easily studied by echography. different organs, it can be seen how tremen- One of the first signs of puberty in girls is dous these changes are. Compared with the the onset of breast development. The physical age of 10 years, the body weight, the anterior characteristics of the stages of breast develop- pituitary and the pancreatic islets about dou- ment have been outlined by Tanner and his ble during this period, the thyroid triples, the group [5,6]. Many clinicians combine breast ovaries increase five-, and the testes even and pubic hair standards into one staging; about 20-fold. however, it seems to be important to stage In the following, first a short account will breast and pubic hair development separately be given of the physical changes of sexual to be able to determine if there is a discrep- maturation and their clinical relevancel. [1]. ancy between the two stages, as can be the In the second part, a short review of aspects of case e.g. in excessive adrenal androgen secre- growth and skeletal maturation will be given tion such as in late-onset congenital adrenal and finally, hormonal changes that take place hyperplasia. Breast development is mainly during pubery will be briefly discussed. regulated by ovarian estrogen secretion, but pubic hair development is stimulated by adrenal androgen secretion. The stages of Female secondary sex characteristics breast development are individually variable: In girls, secondary sexual development some adult women never progress beyond includes the enlargement of the ovaries, stage IV, and some girls progress directly from uterus, vagina, labia, and breasts and the stage III to stage V. Unilateral breast develp- growth of pubic hair [2,3]. The vagina begins ment is normal in early puberty and may to increase in length before the appearance of persist for several months before the other secondary sex characteristics and continues to breast bud appears. Marked or prolonged enlarge until menarche. The cytologic developmental asymmetry, however, may changes of the vagina occur as a result of reflect estrogen insensitivity of the breast tis- estrogen stimulation. The vaginal epithelium sue, prior trauma, or underdevelpment of the undergoes a transformation from parabasal to underlying pectoral muscle. Adolescent girls about 50% intermediate cells. At approximate- are often concerned about breast size, think- ly one year before menarche, the vaginal pH ing their breasts might be either too small or converts from neutral to an acidic environ- too large, but there are no normal standards ment secondary to the presence of lactobacilli. for size. Height, weight, level of estrogen and With puberty the size of the mons pubis progesterone once in the normal adult range, increases by fat accumulation and the labia or breast size of other women in the family majora become larger. The surfaces of the does not have a high correlation with amount labia develop fine wrinkles. With great indi- of breast tissue, but overall non-gladular vidual variation, the clitoris also increases in breast size is somewhat influenced by weight. size [4]. Concerning the uterus, it has no flex- Experience with tall girls treated with ion during childhood. In the prepubertal high doses of estrogens to reduce final adult period, the corpus increases only slightly, height also shows that breast size is not direct- accounting for about one third of the com- ly related to the quantity of circulating bined weight of cervix and corpus. The estrogens, because in these girls breast size is growth of the uterus during early puberty is not larger than in normal untreated girls [7]. primarily caused by the enlargement of the The age at which each stage of breast myometrium. The endometrium does not development occurs has been determined in begin to develop until after the onset of secon- longitudinal studies in American, British and dary sexual characteristics, and rapid increase Swiss girls [3,6,8]. The complete process of 17 Skeletal and Sexual Maturation

breast development, from the appearance of cycles in 12- to 14- year-old girls are anovular, stage II to the attainment of stage V, takes an whereas at the age of 21 years, 60% to 80% of average of 4 years, with a range of 1.5 to 9.0 the cycles are ovular. years. As far as pubic hair is concerned, the Some voice change occurs in pubertal sequence in girls generally begins with the girls, but unlike in boys, may consist more of appearance of breast buds and linear growth an alteration in quality than in pitch [9]. A acceleration but a number of normal girls time plan in girls has been established from develop pubic hair before breast enlargement the Swiss study by Largo and Prader [8]. has occurred. The age at which each stage of pubic hair development occurs has also been determined in longituidinal studies. Marshall Male secondary sex characteristics and Tanner [6] used photographs to study Secondary sexual development in boys pubic hair growth so the ages at which sub- includes genital development, testicular vol- jects were said to reach stage II are somewhat ume, pubic and axillary hair growth and later than in other studies. The mean duration change of voice. The sequence of pubertal of time to progress from stage II to stage V development of the external genitalia in most pubic hair is 2.5 years, with a range of 1.4 to 3. normal boys begins with testicular enlarge- 1 years. There is a high correlation between ment. The changes at puberty in boys are breast and pubic hair stages for black and primarily the result of androgen secretion by white American girls [3]. Black girls are more the testes. Adrenal androgens account, if at advanced than whites for each chronologic all, for a small part of very early pubic hair age and girls in Western regions of the United only. Again, it is better to describe the genital States lagged slightly behind girls from other stage separately from the pubic hair stage. regions in pubertal development. Cross-sectional data of testicular volume Axillary hair occurs at a mean age of 13.1 are not very valuable, because they show a years in American girls, 12.5 years in British very wide distribition. A testicular volume of girls, and somewhere in beween in Swiss girls. about 8ml may still be normal. In this context, Axillary hair takes approximately 15 months it has to be said that the inexperienced exam- from first appearance to adult distribution. iner using Prader's orchiometer generally The apocrine glands of the axilla and vulva obtains too high volumes. Although measure- start to function at about the same time as ment of tesicular volume with this instrument hair appears in these regions. Acne is often a is only a semiquantitative method, it gives major concern for the adolescent girl and quite reproducible results with experienced occurs at an age of around 13 years. examiners. The imporant thing is to subtract Because of its sudden onset and its impli- mentally the scrotal tissues. In this context, it cations, the first menstrual period, or menar- can be said that there is a gap between normal che, has often been regarded as the beginning and subnormal testicular volume: while we of womanhood in ancient cultures. Menarche have never seen a completely normal subject generally occurs within 2 years of the onset of with volumes below 8ml, we also have never breast development and after the peak of the seen a patient with Klinefelter's syndrome growth spurt in height. In a larger study in the with a volume above 5ml. United States the mean age of menarche was Curves of testicular volume from longitu- 12.8 years, with a range of 10 to 16 years. This dinal studies are more useful, also for practi- great variability is mainly due to differences cal purposes [10], because as in growth in bone age. Much variation in the length of curves, the variation is smaller (Fig. 1). The the menstrual cycle and in the amount and augementation of testicular volume, or "tes- duration of flow in otherwise healthy girls in ticular growth velocity" has a peak around the normal. Approximately 90% of all menstrual age of 13.5 years (Fig. 2). Interestingly, this is 18 ZACHMANN

Fig 1. Testicular volume from a recruitment of the Zurich longitudinal study.

Fig 2. Velocity of testicular increment in relation to CA = chronologic age , BA= bone age, PHV= peak height velocity, PH= pubic hair (Tanner stages), LVB5= last volume below 5ml. just before peak height velocity is reached. The age at which each stage of genital development occurs has also been determined from stage II to stage V pubic hair lies in different longitudinal studies [5,11,12]. The between 1.6 and 2.7 years in American, British mean length of time to complete the process of and Swiss boys. There is quite a close concor- genital development, from the appearance of dance between pubic hair and genital stages stage II to the attainment of stage V, ranged in boys and in Americans no difference exists from 3.1 to 3.6 years in American, British and between black and white boys. Swiss boys. The development of axillary and facial Phallic enlargement usually occurs 12 to hair is highly variable, with axillary hair 18 months after testicular enlargement. It is usually appearing about 2 years after pubic difficult to measure the penis accurately in a hair II growth. An interesting phenomenon to child or adolescent and it is preferable to give mention on the side is that development of no precise values. Measuring noctural tumes- pubic and axillary hair is in a still unknown cence of the penis is not technically easy, but way to some extent not only dependent on may be more useful [8]. androgen secretion but also on the presence of Pubic hair generally appears shortly after growth hormone. For a long time clinical genital development has begun [12]. The age experience has shown that hypopituitary at which each stage occurs in American, males may have slow and scant development British and Swiss boys has been compiled by of pubic and axillary hair, either spontaneous- Wheeler [3]. The somewhat later age for stage ly in isolated GH-deficiency or after adminis- II pubic hair development found in the traion of testosterone if there is simultaneous British boys is again most likely not a reality gonadotropin deficiency. We have computed but the result of the photographic technique the total testosterone dose in mg/mm2 that is used. The mean duration of time to progress required to produce axillary hair in males 19 Skeletal and Sexual Maturation

almost over, making it of little use for developmental ratings unless it is studied longitudi- nally [15]. Regarding spermarche, together with the growth of the penis, the seminal vesicles, prostate and bulbourethral glands enlarge and develop. The avearge age of first ejaculation occurs in early to midpuberty and usually precedes the pubertal growth spurt. The mean age for the first conscious ejaculation occurs at age 13.5 to 14.3 years. Breast enlargement in boys at puberty is characterized by an increase in areolar diame- ter and in a large proportion of about one third of all boys by the development of pal- pable and occasionally tender subareolar breast tissue. Pubertal gynecomastia thus is a physiologic phenomenon. It may be unilateral, symmetric or asymmetric and occurs mostly during genital stages Ill and IV and regresses after 12 to 18 months. Occasionally, it does not regress and may increase to necessi- tate medical or surgical treatment [16]. The time plan in boys was again estab- Fig 3. Total testosterone dose to induce axillary hair in lished for Swiss boys by Largo and Prader patients with normal and deificient GH secretion. [12].

Changes in body size and shape and with normal GH-secretion and in GH-deficient aspects of skeletal maturation patients (Fig. 3). This dose is homogeneous in As a mean height distance curve shows, the first group. By contrast, in the GH- prepubertal height is nearly identical in both deficient patients not treated with hGH the sexes with a minimal difference in favor of the mean dose to have this effect is much higher boys. This is caused by a slight difference in and there is a much wider scatter [14]. perinatal growth velocity, probably due to the Development of facial hair begins at a increased testosterone secretion in boys dur- mean age of 14.9 years. The regular sequence ing that period. During puberty, however, of facial-hair growth is the appearance of differences between the sexes show up and pigmented hair at the upper lip, spreading to body maturation is faster in girls than in boys. form a moustache, followed by hair on the This is demonstrated by the difference of cheeks and below the lower lip and finally about 2 years, which is present in the time- over the chin. tables of girls and boys, and by the timing of The male voice change at puberty is the reaching adult height. This difference is result of androgen-induced larynx enlarge- equally expressed by the skeletal maturation, ment. Its timing is also variable but generally which is faster in girls not only at puberty but corresponds to the pubertal growth spurt or long before, even in infancy, and reaches a somewhat later. The voice change is gradual difference of about two years in puberty. and is often not complete until adolescence is Puberty, and this should always be kept in 20 ZACHMANN

mind, begins at a certain stage of skeletal better standardize the valutation. maturation which is identical in both sexes. Together with other centers we have car- This is indicated by the radiological appear- ried out a study on the accuracy of bone age ance of the sesamoid bone of the thumb in estimation by experienced trained technicians both sexes at the same stage of maturation, and radiologists from different centers and namely between the first appearance of the pediatric endocrinologist consultants [19]. secondary sex characteristics and the peak of The mean bone age according to Greulich and the pubertal growth spurt. Thus, what we call Pyle obtained by different examiners has been a bone age of 11 in girls and 13 in boys is established. Since the patient population and exactly the same maturational stage. The rea- their bone age values do not represent a Gaus- son why bone maturation in girls is faster than sian distribution no standard deviations but in boys before puberty is unknown and does the ranges of the obtained values were indicat- not seem to depend on the sex hormone secre- ed. The values obtained by the different tion. This sex difference in the speed of body examiners were very similar. If the mean maturation is still after many studies one of value of all four estimations is considered to the least understood problems in the research be the "true" bone age the difference of this in this field. value from that of one examiner can be con- Since girls mature faster their height ex- sidered as an indication of the quality of his pressed as a percentage of adult height is rating. The mean differences of the values of greater han that of boys during the whole each examiner from the mean value of all period of growth. Looked at it in this way, examiners, which have been calculated both sexes reach the milestones of develop- disregarding the plus or minus signs, do not ment at the same moments. Minimal prespurt exceed 0.5 years in any group. Also the ratings height velocity (MHV) is reached at 82.5% of according to Tanner et al. were very similar. adult height, the sesamoid bone appears at The mean differences of the values of each 89%, and peak height velocity (PHV) is rea- examiner from the mean values of all three ched at 90% of adult height. In addition, examiners were smaller than those observed menarche in girls occurs at 95.3%, which with the method of Greulich and Pyle, and did allows a mean postmenarcheal growth of 7. not exceed 0.3 years in any group. 8cm. These are, of course, only mean values The ratings according to Tanner et al. and postmenarcheal growth may very from 3 are always somewhat higher than those to 11cm. according to Greulich and Pyle. The mean Concerning skeletal or bone age, it is differences between the two methods, taking important to know that the degree of skeletal the plus or minus signs into account, are +0. development is a reflection of the degree of 4 years in the boys and +0.3 years in the girls. physiologic maturation of a subject. In addi- The plus sign means that the Tanner et al. tion, the determination of skeletal age will values are higher than those of Greulich and indicate how much growth remains for a child Pyle. The mean intraobserver errors, or the and allows the prediction of final height. The replicability, which was studied in a series of Greulich and Pyle atlas is most frequently radiographies was 0.2 years and compares used [17], but depending on the situation, it is favorably with those observed by others. more useful to use the more precise method of The problem of height prediction needs Tanner et al. [18]. Very recently, Tanner and some further comment. There are several coworkers have computerized this system; we methods available and three of them are in have had an opportunity to test it thoroughly. general use: those according to Bayley and This system cannot yet be recommended for Pinneau [20] and Roche et al. [21], both general use and it does not save time for the based on bone age according to Greulich and experienced examiner, but it might help to Pyle, and that of Tanner et al. [18], based on 21 Skeletal and Sexual Maturation

Fig 4. Deviation of height prediction from adult height with different methods in normal subjects. BP= Bayley & Pinneau, RWT= Roche, Wainer and Thissen, T= Tanner et al.

bone age according to the same authors. The normal bone age [23]. In excessively tall chil- Bayley and Pinneau method is classic and well dren the method of Bailey and Pinneau tends known. It is relatively simple to perform. This to overestimate slightly by a mean of 2.3cm. is an advantage and disadvantage at the same On the other hand, the method of Bayley and time: advantage, because it saves time, and Pinneau is much more accurate in children disadvantage, because people who are not who have true growth disorders and an abnor- experienced with the method tend to do it in a mal bone age deviation from chronologic age, sloppy way with large errors. The Roche and such as children with precocious puberty, Tanner Mark I methods take midparent where the other two methods tend to grossly height into account and are somewhat more overestimate (Fig. 5). complicated to use. It is best and easiest to do The standard deviation of the prediction the calculations by computer. We have devel- in control groups is nearly 4cm, which indi- oped, together with the Ares-Serono company, cates that while the mean prediction in a such a system which is called TGE (theoretical group is very accurate the individual predic- growth evaluation, [22]). tion has a relatively large error. This is well In our experience (Fig. 4) the three known and has been discussed in the original methods are quite reliable in children who presentations of the various methods. have no growth disorder and a more or less Another consequence of pubertal growth 22 ZACHMANN

Fig 5. Deviation of height prediction from adult height with different methods in patients with advanced bone age and precocious puberty. Note the grossly overestimating values with the RWT and T methods.

needs to be considered: what is the cause of ered an important factor. the sex difference of adult height? In various Everybody knows that normal adult studies a mean difference of stature between height is not reached - at least not in due time men and women of 10 to 14cm has been found. - when growth hormone , thyroxin or insulin is At first glace one would tend to believe that it deficient. By contrast, the sex hormones do is due to the different effects of the sex hor- not have any influence on adult height. mones and that testosterone has a stronger Untreated XY-individuals with deficient effect than estradiol. This hypothesis has, gonadal function have no pubertal spurt but however, been shown to be wrong. On the continue to grow slowly over a prolonged contrary, the sex difference of adult height is period and reach normal adult height. already programmed in early childhood as Equally, XX-individuals, e.g. with pure indicated by the faster skeletal maturation of gonadal dysgenesis, reach normal adult girls in the presence of an identical growth height. The sex steroids do, however, acceler- velocity in both sexes during the period ate growth and skeletal maturation if present between infancy and puberty. There is no too early or in excessive quantities, such as in clear explanation for this. In animal experi- untreated congenital adrenal hyperplasia or ments the possibility of perinatal imprinting precocious puberty. of the male pattern by testosterone is consid- In contrast, the adolescent growth spurt 23 Skeletal and Sexual Maturation

Fig 6. Spontaneous pubertal growth spurt in androgen resistance.

clearly and evidently depends on gonadal adrenal androgens. steroids. It thus can be said that sex hormones The sex hormones thus modulate the cause the pubertal growth spurt. Studies on body proportions, but have no influence on the pubertal growth spurt have only been adult height. This statement, which may be carried out quite recently and it may be that surprising to some not familiar with the in earlier times it was absent or smaller due to details of growth analysis, is confirmed by the nutritional or other unfavorable factors [24]. results of the Zurich longitudinal study: in Often it is still said in textbooks that adrenal normal children, adult height is independent androgens play a more important role in the of the timing and intensity of the pubertal pubertal growth spurt of girls than estrogens. growth spurt. Adult height and velocity of This is not true in our experience. Our studies maturation therefore are independent in patients with androgen resistance (Fig. 6) multifactorial variables [26]. In the Zurich show that estrogens alone are capable of in- longitudinal study, a sex difference in adult ducing a pubertal growth spurt in the absence height of 12.5cm was found. Formal analysis of androgen action [25]. A prerequisite is, of the components gave the following however, normal GH-secretion. Patients with results: +1.5cm in boys are caused by more the classic type of androgen insensitivity have prepubertal growth, which occurs in the peri- a pubertal growth spurt equal in magnitude to natal period. +6.5cm are due to the delay of that in normal girls. From this observation it the spurt in boys, +6cm are caused by the may be concluded that also in normal girls the higher peak in boys, and -1.5cm are ex- pubertal spurt is exclusively due to ovarian plained by more post-spurt growth in girls. estrogens together with GH, and not to It is convenient to define the timing of the 24 ZACHMANN

Fig 8. Mean course of pubertal development in girls.

Fig 7. Mean course of pubertal development in boys. MHV= minimal prepubertal height velocity, PHV long legs (SILH or subischial leg height), rela- = peak height velocity , 99%AH= 99 percent of tively short sitting height (SH) and a relatively adult height reached. still smaller head circumference (HC). The data show that otherwise normal children adolescent growth spurt by the following with familial tall stature are indeed relatively milestones: starting point is the age of mini- more long-legged than children whose height mal prespurt height velocity (MHV). The is within normal limits [28]. Because pubertal peak is reached at the age of peak height children during the early growth spurt always velocity (PHV), and the spurt ends at the age, have relatively long legs this difference can when adult stature is attained. Since this last only be appreciated by comparing SDS- point cannot be identified with precision values. The results also show that the weight because of its asymptotic nature, it was decid- in tall children is generally relatively low and ed to select the age, when 99% of adult height that their heads are relatively small. Weight is completed (99AH, Figs. 7 and 8). In spite of gain is regulated by factors other than those the sex difference in chronologic age, the regulating growth. While tall normal children pattern of sexual maturation is also identical are known to secrete more GH and IGF I in both sexes. than short normal children, the pubertal Also the body proportions change during weight gain depends not on these factors, but puberty: the legs accelerate in growth first, rather on the quantities of secreted sex hor- followed by the spurt in trunk length almost 1 mones, which are not higher in tall than in year later [27]. Leg growth is not uniform: the short normal children. As far as the relatively foot accelerates first, followed by the calf and small head circumference is concerned, this is thighs. Similarly, the growth spurt of the also biologically understandable: gowth of the hand and forearm precedes that of the upper skull is only to a small extent regulated by the arm. Thus the more distal parts of the limb usual growth factors, but much more by the increase their growth rate before the proximal growth of the brain, which follows other rules. parts. Of course, these changes are taken into This also explains why in children with short account when the proportions are calculated stature the opposite is found: their head cir- in SDS or standard deviation scores for sitting cumference is relatively large compared to height and subischial leg height. Interestingly, their short stature. when studying boys and girls with familial tall In addition to the maturation of secon- stature we have seen that they have relatively dary sexual characteristics puberty is also 25 Skeletal and Sexual Maturation

characterized by marked changes in body teristic sexual dimorphism seen in young composition. Lean body mass, primarily re- adults. Broadening of the shoulders relative to flecting muscle mass, begins to increase dur- the hips is characteristic of males, whereas ing early puberty in both boys and girls. In broadening of the hips occurs in females. girls, muscle mass peaks at menarche and Relative development of these proportional then diminishes. In boys, lean body mass differences becomes evident when the ratio continues to increase throughout puberty, between biacromial and bicristal breadth is attaining higher levels than in girls. In boys compared. Between 9 and 18 years, the ratio in there is a marked increase in width of bone boys is almost constant (about 1.37), whereas and muscle during adolescence, usually ac- it steadily decreases in girls (from 1.35 to 1.27), companied by a loss of fat. In addtion to because they develop broader hips relative to lengthening, bones also grow in thickness their shoulders. [29-32]. During puberty, the cortical layer of The growth of the head and face is nearly bone grows more in boys than in girls. The complete in childhood. Changes in the size muscle widths of boys and girls show maximal and shape of the head and face are, however, velocities coincident with or slightly after apparent during puberty. The greatest growth PHV. In females, lean body mass increases is in the mandible. As a result of this, the more gradually. The accurate measurement of profile becomes straighter, and the chin more lean body mass is difficult and cannot be used pointed. for practical purposes, but measurement of electrical impedance can give an approximate estimation of fat percentage and thus also of Hormonal changes during puberty lean body mass. The basal gonadotropin levels increase The decreasing contribution of muscle during puberty [33-36]. FSH increases more in mass to body weight in girls during adoles- girls, while LH is about the same in both cence and into young adulthood reflects accu- sexes. Basal gonadotropins are, however, of mulation of fat. Fat mass increases during late little practical value in assessing the pubertal puberty in girls and by the time maturation is status. They are of diagnostic importance in complete the total amount of body fat in girls the presence of primary hypogonadism but in is almost twice that in boys. In boys, the upper other situations a LHRH-test has to be perfor- arm, calf, and thigh all show a loss of fat, med. Some of the time-related phenomena of whereas girls lose fat only from the upper gonadotropin secretion are difficult to study arm, although there is a slowing of fat accu- in the human and have been better studied in mulation on the thigh and calf. primates [37]. More important than absolute The amount and distribution of water values is the advent of a rhythmic, pulsatile also change at adolescence. The contribution secretion of gonadotropins in boys [38] and of water to body weight increases from 61 to girls [39,40]. Only at advanced stages do the 65% in boys and decreases from 61 to 54% in characteristic midcycle peaks appear, which girls between 12 and 17 years of age. The are a prerequisite of ovulation. extracellular water component of total body Of course the most obvious hormonal water is stable in both sexes (25% to 26%), changes during puberty are the markedly whereas the intracellular water component increasing secretion rates of sex steroids [41]. increases in boys (from 36% to 39%) and Oestrogens in girls (Fig. 9) increase very steep- decreases in girls (from 36% to 29%). ly as a consequence of the increased Changes of proportions other than those gonadotropin stimulation [42] and also the related to height are minor during the acute response to exogenous hMG becomes preadolescent years. Sex differences in the more marked [43]. Estradiol, which predomi- adolescent growth spurt produce the charac- nantly originates from the ovaries increases 26 ZACHMANN

Fig 9. Plasma oestrone and oestradiol in girls during the first months of life and in relation to pubery stages. Fig 10. Plasma testosterone in boys in relation to pubic hair stages. considerably more than estrone, about 60 percent of which is produced in the adrenals. Our results in subjects with high and low When evaluating estradiol levels, they should testosterone levels indicate that salivary tes- be related to bone age and/or pubertal stages tosterone in boys correlates well with total rather than to chronologic age, because plasma testosterone (Fig. 11). Since salivary related to the latter the variation is immense. testosterone predominantly reflects free testo- The same is true for testosterone in boys sterone in plasma, and since in metabolically (Fig. 10), which increases steeply [44]. Not normal boys and adolescents free testosterone only the basal values but also the response to corresponds to only a small percentage of total exogenous hCG, reflecting the secretory plasma testosterone, the quantity of testoster- reserve of the Leydig cells, are higher than one in saliva is, however, small. The method is before puberty [45]. These values also have to thus suitable to monitor testosterone secretion be related to pubertal stages rather than to in male adolescents, but not in prepubertal chronologic age. In some cases with disorders boys or in girls. of puberty it would be usuful to monitor tes- The role of DHEA and its sulphate tosterone levels regularly at weekly or even DHEAS from the adrenals in pubertal devel- daily intervals; the values might have implica- opment is still unclear. They start to increase tions for later fertility [46]. Since this is diffi- at the time of adrenarche, much earlier than cult to do in plasma, others [47] and we [48] the onset of puberty. They increase more have developed a method for the estimation of slowly than gonadal estradiol and testoster- salivary testosterone which is quite reliable. one. Probably the role of DHEA for puberty is 27 Skeletal and Sexual Maturation

Fig 12. Effect of acutely administered testosterone propionate (15 mg/mm2.day i.m. for 6 days) on Fig 11. Correlation between total plasma testosterone 15N-balance in a GH-deficient patient off and on (TPT) and salivary testosterone (ST). long-term hGH treatment.

not very important because, for example, have probably not only such indirect effects, patients with Addison's disease and low but also more direct effects through their DHEA generally have normal pubertal devel- anabolic action. The synergistic metabolic opment with the exception of scant pubic hair effects between GH and sex hormones are in girls. physiologically interestig and also of practical Of course not only sex steroids but also importance. Many classic metabolic studies growth hormone [49-55] and its binding pro- indicate that both hGH and testosterone are tein [56,57], as well as IGF I [58-61] and its potent nitrogen retaining agents [67]. Such binding protein 3, are higher during puberty studies have shown that the nitrogen retention than during prepuberty [62-64] and act after hGH in small doses is more marked in together with the sex steroids in causing the GH-deficient than in normal subjects. On the pubertal growth spurt and body changes [65, other hand, testosterone given in appropriate 66]. Although there is a wide variation, there doses for 5 to 6 days causes nitrogen retention is a considerable increment of IGF I during in all subjects with the exception of patients this period. The difficulty with IGF I is, with the classic type of androgen insensitivity however, that its serum levels do not always [68]. Compunds containing the stable, non- reflect the overall production in the liver and radioactive isotope 15N are suitable for studies peripheral tissues. of the nitrogen and protein metabolism in How do these hormones work together children, and we have carried out a number of with the sex hormones? The fact that sex studies in different types of conditions. steroids have a priming or stimulating effect The examples of two male adolescents on GH secretion supports the concept that with GH-deficiency show these interrelations. they also stimulate GH-secretion under physi- They were studied in the following way: in the ological pubertal conditions, which in turn first patient, the response to testosterone was stimulates the production of IGF I. Another tested once without any additional treatment, effect of GH is probably the stimulation of and once on hGH, which had been started androgen receptors. However, the steroids three months earlier. In the second patient, 28 ZACHMANN

Fig 13. Growth-stimulating effect of testosterine in patients with normal GH-secretion and in patients with GH-deficiency on and off hGH-treatment.

the opposite was done, and his response to does not influence or even reduce the acute acute hGH was tested once without additional response to hGH. treatment, and once on testosterone, which This interrelation also has a practical had been started three months earlier [69]. In aspect concerning the growth-promoting the first patient, the balance change induced action of testosterone and its relation to GH. by testosterone alone was smaller than in The growth response of GH-deficient patients normal boys. When the test was repeated on to androgens or anabolic steroids varies con- long-term hGH, the balance change became siderably individually. In the majority of considerably higher than without hGH or in GH-deficient patients, however, the growth normal subjects (Fig. 12). In the second response to androgens alone is considerably patient, the response to hGH alone was more smaller than that in boys with normal GH- marked than in normal subjects, as expected secretion. We studied the effect of exogenous in GH-deficiency. When the test was repeated testosterone in comparable doses in patients on long-term testosterone, the basal balance with normal GH-secretion (isolated increased, but the response to acutely ad- gonadotropin deficiency and anorchia) and in ministered hGH was reduced. From these GH-deficient patients [70]. Among the latter, observations the following conclusions may be one group was treated with testosterone alone drawn: in the absence of GH, 1) long-term and another one simultaneously with hGH hGH has no influence on the basal 15N- (Fig. 13). In the patients with normal GH- balance, but enhances the acute response to secretion, there was a marked growth response testosterone markedly and 2) long-term testos- to testosterone. In the GH-deficient patients terone increases the basal 15N-balance but off hGH there was a poor response, but in 29 Skeletal and Sexual Maturation

those treated with hGH the response was 7. Zachmann M, Ferrandez A, Murset G, equal to that in the patients with normal Prader A, Estrogen treatment of excessive- GH-secretion. From this it may be concluded, ly tall girls. Hely paediatr Acta 1975; 30: that under physiological conditions also, tes- 11-30. tosterone can only have its stimulating effect 8. Largo RH, Prader A, Pubertal develop- on the growth spurt when GH and IGF I are ment in Swiss girls. Hely paediatr Acta present in sufficient quantity. The situation is 1983; 38: 229-43. less clear in girls, but it seems that they also 9. Pedersen MF, Moller S, Krabbe S, Bennett need estradiol, GH and IGF I for a normal P, Svenstrup B, Fundamental voice fre- pubertal spurt. quency in female puberty measured with In this brief overview it has been only electroglottography during continuous possible to give a general impression on some speech as a secondary sex characteristic. A somatic and hormonal aspects of this most comparison between voice, pubertal interesting and eventful period in life, which stages, oestrogens and androgens. Int J we call puberty. We know much about it, but Pediatr otorhinolaryngol 1990; 20: 17-24. we still don't know how it is trigered and what 10. Zachmann M, Prader A, Kind HP, Hafliger are the original laws of nature that give it the H, Budliger H, Testicular volume during starting signal [71]. But we are learning fast adolescence. Cross-sectional and longitudi- and experiments at the level of the hypoth- nal studies. Hely paediatr Acta 1974; 29: alamic neurotransmitters are beginning to 61-72. shed some light on this secret [72,73]. When 11. Marshall WA, Tanner JM, Variation in the working in this area, ever more new and inter- pattern of pubertal changes in boys. Arch esting aspects are arising. To finish, the fol- Dis Child 1970; 45: 13-23. lowing saying of Solon, the old Greek philoso- 12. Largo RH, Prader A, Pubertal develop- pher, seems to be appropriate. He said: "I ment in Swiss boys. Hely paediatr Acta grow older ever learning many things". 1983; 38: 211-28. 13. Horita H, Kumamoto Y, Aoki M, Yamagu- chi Y, Satoh Y, Suzuki N, et al. Clinical References significance and usefulness of measuring nocturnal penile tumescence in children. 1. Van Vliet G, Clinical aspects of normal Nippon Hinyokika Gakkai Zasshi 1991; 82: pubertal development. Horm Res 1991; 36: 1939-46. 93-6. 14. Zachmann M, Prader A, Anabolic and 2. Barnes HV, Physical growth and develop- androgenic effect of testosterone in sexu- ment during puberty. Med Clin N Amer ally immature boys and its dependency on 1975; 59: 1305-17. growth hormone. J Clin Endocrinol Metab 3. Wheeler MD, Physical Changes of Puberty. 1970; 30: 85-95. Endocrinol Metab N Amer 1991; 20: 1-14. 15. Saida H, Okamoto M, Imaizumi S, Hirose 4. Sane K, Pescovitz OH, The clitoral index: a H, A study of voice mutation and physical determination of clitoral size in normal growth-- a longitudinal observation. girls and in girls with abnormal sexual Nippon Jibiinkoka Gakkai Kaiho 1990; 93: development. J Pediatr 1992; 120: 264-6. 596-605. 5. Tanner JM, Growth at adolescence, edition 16. Zachmann M, Eiholzer U, Muritano M, 2. Oxford: Blackwell Scientific Publica- Werder EA, Manella B, Treatment of tions, 1962. pubertal gynaecomastia with testolactone. 6. Marshall WA, Tanner JM, Variations in Acta Endocrinol (Kbh) 1986; 112: Suppl. the pattern of pubertal changes in girls. 279, 218-26. Arch Dis Child 1969; 44: 291-303. 17. Greulich WW, Pyle SI, Radiographic atlas 30 ZACHMANN

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