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Literature reviews
Adolesc Pediatr Gynecol (1993) 6:47-52
Adolescent and Pediatric Gynecology © 1993 Springer-Verlag New York Inc.
Literature Reviews Reproductive Endocrinology The Use of Antiandrogens in the Treatment of Benign Hirsutism in the Adolescent Reviewed by: Carol J. Levi, M.D. and Marvin L. Swanson, M.D., Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Sinai Hospital of Baltimore, Baltimore, MD. Antiandrogens are substances that prevent androgens from expressing their activity at target sites. These substances block androgenic activity in several ways: they modulate enzymatic activity, competitively bind to androgen receptors or testosterone-binding globulins, and/or increase aromatase activity.1,2 Interest in these compounds has grown recently regarding their use in hyperandrogenesis syndromes in females (e.g., hirsutism, polycystic ovarian disease (PCO), and acne). They have demonstrated promise in treating benign hirsutism. In a prior issue, we have reviewed the current data on oral contraceptive (OC) therapy for hirsutism in adolescents. This article will continue to examine therapies for benign hirsutism in adolescents, specifically focusing on antiandrogen therapies, and the new data emerging on this topic. Adolescents who present with benign hirsutism are a unique group in that they have specific concerns regarding self-image. OCs were examined in our previous review as an excellent, although not necessarily the only, treatment for benign adolescent hirsutism. OCs generally represent a good first line treatment for mild-to-moderate hirsutism in adolescents with the added benefit of providing contraception. They are packaged easily for the relatively noncompliant adolescent and have relatively few side effects. However, OCs are not always adequate treatment for the more severely affected benign hirsute patients, and other therapies may be needed. Antiandrogens, with or without OCs, may provide a more effective treatment in the adolescent with severe hirsutism. Five currently studied antiandrogens will be reviewed in this article: cyproterone acetate (CA), spironolactone (SP), flutamide (FL), cimetidine (CI), and ketoconazole (KC). CA and SP retain the steroid backbone common to estrogen and androgens, whereas FL, CI, and KC are nonsteroidal compounds. It should be remembered
that no medications are currently specifically approved by the FDA for the treatment of hirsutism. CA was originally developed as a progestin. It was found to have antiandrogenic activity because it caused feminization of male rat fetuses. It is a steroid antiandrogen that inhibits the formation of the nuclear androgen-receptor complex, and thus is primarily a competitive inhibitor of androgens. By decreasing levels of sex hormone binding globulin (SHBG), free serum testosterone (T) increases, which in turn increases its metabolic clearance rate. 3 In addition to being a potent progestin and an antiandrogen, it is also a weak glucocorticoid. CA is commercially available only in Canada and Europe. It is supplied in SO-mg doses or in combination with SO mcg or 3S mcg ethinyl estradiol as an OC in a 2-mg dose. The higher doses of CA are generally used to treat hirsutism. Because of CA's long half-life, typically 2S-100 mg can be given in the first 10--11 days of a birth control pill cycle. Side-effects of high-dose CA are similar to those of OCs in general, except that there seems to be more breast tenderness, amenorrhea, and weight gain with high-dose CA. As a treatment for hirsutism, it has been shown to significantly decrease hirsutism with single i.m. doses of 300 mg q month x 6 months which minimizes the side-effects of breast tenderness, amenorrhea, and weight gain. 3 ,4 Topical application of CA is ineffective. 2 Its use in a 2.0-mg dose in combination with ethinyl estradiol has been shown over a period of 3--6 months to significantly increase SHBG and decrease dehydroepiandrosterone sulfate (DHEA-S).4 Another study did not find a decrease in DHEA-S, suggesting that adrenal suppressive effects were unlikely. 3 The effect on CA in SHBG seems to be dose related, with higher doses resulting in decreased SHBG and the 2-mg dose resulting in increased SHBG. In any event, most of the patients noticed improvement in hirsutism between the eighth and twelfth cycle of treatment. Although it apparently will do well for the adolescent hirsute population, it awaits FDA approval for treatment in the U.S. and is currently not available. Spironolactone has been studied frequently in adults,5-8 but in relatively few adolescents. It has a steroid backbone structure, and acts as an aldosterone antagonist with antiandrogenic effects. AI-
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Literature Reviews
though the precise mechanism of antiandrogen action of SP is beyond the scope of this article, in general, SP decreases serum T, progesterone, and follicle-stimulating hormone (FSH) while increasing estradiol. 7 It inhibits the production of T by decreasing the microsomal cytochrome P450dependent enzymes. I Additionally, SP competes for androgen receptor binding sites, thus decreasing serum T levels. Three studies 6,8,9 found that serum androgens were not helpful in predicting the clinical response with SP in hirsute patients, suggesting that peripheral androgen metabolism is the major determinant of hirsutism in women. SP is equally effective in the treatment of idiopathic hirsutism and hirsutism associated with PCO. 8 Long-term therapy is important because the duration of therapy is directly related to clinical response. When it is administered at 100-200 mg/day (in two divided doses), facial hair became less coarse and growth occurred at a decreased rate. 7,10 It is important to realize that SP produces diuresis, decreases blood pressure, increases insulin levels,l1 as well as predisposes to menstrual irregularities. The menstrual irregularities result from the drug's effect on enzyme activity in the steroid pathways, as well as actions at the androgen and estrogen receptors. Adolescents may not tolerate these side effects and compliance may become an issue. Cyclic administration of SP may minimize side effects without decreasing efficacy, although this remains controversiaC,12 Prior characteristics of the menstrual cycle largely influence the incidence of menstrual irregularities. 13 OCs used in combination with SP will regulate potentially irregular cycles, and avoid this side effect. Although not considered to be a carcinogen,14 SP is a potential teratogen. Because it can prevent normal masculinization of a male fetus, its use in the adolescent population should probably be restricted to a combination with OCs to prevent pregnancy. Wild et al. II pointed out that the metabolic consequences of SP/OC treatment should be considered with respect to triglyceride and insulin levels and contraception. OCs tend to increase triglyceride levels whereas SP tends to increase insulin resistance. Insulin resistance appears to be at the root of a syndrome that predisposes to a cluster of metabolic disorders including atherosclerotic cardiovascular disease. 15 When OC/SP are used in combination, factors for cardiovascular disease increase. SP combined with an OC has been shown to improve hirsutism in women with irregular menses and other clinical features of PCO. This improvement was greater than when SP was used alone.23 Interestingly, SP has been tried as a topical therapy with fewer side effects. 12 Its use for acne was ef-
fective; however, its failure in the treatment of hirsutism attests to the importance of systemic levels of the drug which are not achieved topically. Flutamide (FL) is an anti androgen without the steroid backbone, therefore, it carries no glucocorticoid, progestational, androgenic, or estrogenic activities. 16 It has the ability to competitively block androgen action at the target tissue androgen receptor sites without decreasing the serum T level. Two studies evaluated the use of FL in hirsutism. In one study,16 it was administered in a dose of 250 mg b.i.d. in combination with ethinyl estradiol 35 mcg and norethindrone 1 mg with "good results," even in patients who had failed SP treatment. Adequate response required 6-12 months of combination therapy. The most common side effects included dry skin (70%), hot flushes (27%), and increased appetite (25%) although weight gain was not reported. None of the study subjects dropped out of this clinical trial because of side effects. In a more recently published clinical trial,17 FL was given alone in a dose of 250 mg Li.d. to nine hirsute women for 3 months. The FerrimanGallwey scores of assessment of body hair growth in women were significantly improved. Side effects included a high incidence of oligomenorrhea, and a low incidence of dry skin. No other serious side effects were reported. Interestingly, hormonal analysis of the study subjects showed no changes in serum androgens, or in the measured or calculated T fractions, suggesting that FL acts only at the androgen receptor level. This is different than the two other antiandrogens, CA and SP, both of which change the serum androgen levels as previously discussed. This medication may have promise in adolescent hirsutism. More studies will be forthcoming. Cimetidine, a histamine receptor type 2 blocker, has been used as therapy for hirsutism. It blocks the action of androgens at the hair follicle by inhibiting the binding of dihydrotestosterone to androgen receptors. However, earlier reports of success have been dampened by more recent reports of it failures,18.19 and appears not to be effective clinically. Ketoconazole, an antimycotic agent, has also been shown to reduce serum levels ofT, DHEA-S, and androstenedione,zo However, clinical efficacy has been mixed 21 .22 and side effects of mastodynia, gastrointestinal upset, and somnolence may limit its use. Clinical Correlation. In summary, the treatment of hirsutism recalcitrant to oral contraceptives should begin with SP. SP use should be monitored with respect to its side effects, and concomitant contraception is required with its use. Although CA appears to be very effective against
Literature Reviews
hirsutism, it is not yet commercially available in the U.S. Flutamide, although a promising antiandrogen, requires further long-term evaluation before its use is widespread. The hirsute adolescent may require prolonged therapy to successfully decrease unwanted hair growth. A balance between aggressive treatment and multiple side effects will continue to be sought for optimum care of this unique patient population. References 1. Sciarra F, Toscano V, Concolino G, Di Silverio F: Antiandrogens: clinical applications. J Steroid Molec Bioi 1990; 37:349 2. Neumann F, Topert M: Pharmacology of antiandrogens. J Steroid Biochem 1986; 25:885 3. Marcondes JAM, Wajchenberg, BL, Abujamra AC, et al: Monthly cyproterone acetate in the treatment of hirsute women: clinical and laboratory effects. Fertil Steril 1990; 53:40. 4. Prelevic GM, Wurzburger MI, Balint-Peric L, et al: Effects of a low-dose estrogen-antiandrogen combination (Diane-35) on clinical signs of androgenization, hormone profile and ovarian size in patients with polycystic ovary syndrome. Gynecol Endocrinol 1989; 3:269 5. Vetr M: Hirsutism-a low dose spironolactone therapy. Acta Univ Palacki Olomuc Fac Med 1989; 122 6. Spandri P, Gangemi M, Nardelli GB, et al: Testosterone, 17KS, 1713 E 2 , FSH-LH variations and hirsutism modifications during spironolactone therapy. Clin Exp Obst Gyn 1984; 1-2:49 7. Shapiro G, Evron S: A novel use of spironolactone: treatment of hirsutism. J Clin Endocrinol Metab 1980; 51:429 8. Evans DJ, Burke CW: Spironolactone in the treatment of idiopathic hirsutism and the polycystic ovary syndrome. J Royal Soc Med 1986; 79:451 9. Carmina E, Lobo RA: Peripheral androgen blockade versus glandular androgen suppression in the treatment of hirsutism. Obstet Gynecol 1991; 78:845 10. Barth JH, Cherry CA, Wojnarowska F, et al: Spironolactone in an effective and well-tolerated systemic antiandrogen therapy for hirsute women. J Clin Endocrinol Metab 1989; 68-5:966 11. Wild RA, Demers LM, Applebaum-Bowden D, et al: Hirsutism: metabolic effects of two commonly used oral contraceptives and spironolactone. Contraception 1991; 44: 113 12. Messina M, Manier C, Musso MC, et al: Oral and topical spironolactone therapies in skin androgenization. Panminerva Med 1990; 32:49 13. Helfer EL, Miller JL, Rose LI: Side effects of spironolactone therapy in the hirsute woman. J Clin Endocrinol Metab 1988; 66-1 :208 14. Lumb G, Newberne P, Rust JH, et al: 1978 Effect in animals of chronic administration of spironolactone: a review. In: Aldosterone antagonists in Clinical
15.
16. 17. 18.
19. 20.
21.
22. 23.
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Medicine. Edited by Addison GM, Asmussen NW, Corvol P, Kloppenborg PWC, Norman M, Schroder R, Robertson nc. Amsterdam, Oxford, Excerpta Medica, p 132 DeFronzo RA, Ferrannini E: Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991; 14: 173 Cusan L, Dupont A, Belanger A, et al: Treatment of hirsutism with the pure antiandrogen flutamide. J Am Acad Derm 1990; 23:462 Marcondes JAM, Minnani SL, Luthold WW, et al: Treatment of hirsutism in women with flutamide. Fertil Steril 1992; 57:543 Lissak A, Sorokin Y, Calderon I, et al: Treatment of hirsutism with cimetidine: a prospective randomized controlled trial. Fertil Steril 1989; 51 :247 Golditch 1M, Price VH: Treatment of hirsutism with cimetidine. Obstet Gynecol 1990; 75:911 DePedrini P, Tommaselli A, Spano G, et al: Clinical and hormonal effects of ketoconazole on hirsutism in women. Int J Tissue Reac 1988; 3:193 Conget n, Halperin I, Ferrer J, et al: Evaluation of clinical and hormonal effects of hirsute women treated with ketoconazole. J Endocrinol Invest 1990; 13:867 So nino N, Scaroni C, Biason A, et al: Low-dose ketoconazole treatment in hirsute women. J Endocrinol Invest 1990; 13:35 Rittmaster RS: Evaluation and treatment of hirsutism. Fertil Reprod CI N Am 1991; 2:511
Molecular Biology Evidence for a Partial Deletion in the Androgen Receptor Gene in a Phenotypic Male with Azoospermia. Akin JW, Behzadian A, Tho SPT, McDonough PG. Am J Obstet Gynecol 1991;165:1891 and Point Mutation in the DNA Binding Domain of the Androgen Receptor in Two Families with Reifenstein Syndrome. Klocker H, Kaspar F, Eberle J, Uberreiter S, Radmayr C, Bartsch G. Am J Hum Genet 1992;50: 1318. Reviewed by: Leo Plouffe, Jr., M.D., C.M., Section of Reproductive Endocrinology, Genetics and Infertility, Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA. We have previously reviewed the initial report about the localization of the androgen receptor (AR) gene.! The locus, at the Xql1-12 region, has been the subject of intense research since the report by Lubahn et al. 2 Many investigators have described mutations in the AR gene associated with complete androgen receptor insensitivity. 3-5 The two current reports highlight a similar type of gene
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Literature Reviews
mutation associated with partial androgen receptor defects. Several authors over the years have proposed that azoospermia and oligospermia may result from defects in the androgen receptor. Picking up on this idea, Akin et al. studied seven otherwise normal males who presented with azoospermia. One of these seven men was found to have a partial gene deletion in exon 4, a region responsible for DNA binding. This was visible through both polymerase chain reaction (PCR) and Southern blot analysis. Unfortunately, these authors did not present family studies to confirm the association of this mutation with azoospermia. Ideally, one would like to find a similar mutation in the patient's mother and other affected males within the family. Following these studies, the exact nature of the mutation should be determined. Nonetheless, this constitutes one of the first reports of a mutation in the AR gene associated with a very limited phenotypic manifestation. The report by Klocker et al. is impressive for both the breadth of the patients studied and the thoroughness of their methodology. These authors present reports on two families. However, they studied a total of 41 inter-sex patients. Of this group, they identified five study subjects who presented with perioneoscrotal hypospadias and undescended testes, a phenotype compatible with Refenstein syndrome. Three siblings from one family were studied, along with their mother, sister, one aunt, and one uncle. Two affected males from another family were studied along with blood from their mother. Genital skin fibroblasts were obtained from all patients and were studied for specific binding of dihydrotestosterone, 5-a reductase activity. Both were within normal range. The defect they identified is in exon 3, again part of the DNA binding domain of the androgen receptor. The mutation consists of a guanine to adenine substitution at nucleotide 2314. This produces the substitution of threonine for alanine in a zinc finger region of the AR. As a result of this mutation, androgen binding to its receptor is not affected but the activity of the androgen-receptor complex is diminished. A male phenotype does result but undermasculinization is evident. The family information confirms the X-linked nature of this disorder, with the demonstration of the same mutation in the mother and sister of one family. For the molecular biologists among us, the technique utilized to rapidly screen for the presence of the mutation is of particular interest. It involves both PCR and restriction-analysis method. This rapid methodology is particularly suited for clinical applications and is quickly gaining popularity.
Clinical Correlation. The two papers reviewed here point to the varied phenotypes that can be associated with defects in the AR gene. This is now a good model for a genetic disorder in reproductive medicine. One should consider such a defect whether dealing with a child with ambiguous genitalia, a male teenager with pubertal delay, a female with the phenotype of complete androgen insensitivity, or a male with azoospermia. Could it also be that males with cryptorchidism or oligospermia also carry minor mutations in the AR gene? Only time will tell. We can also speculate that some mutations may result in overexpression of androgen action, as has recently been described for McCune-Albright disease and cGMP. In this context, we may gain new insights into the complex problems of hirsutism and masculinization in the female. As a final note, we would like to acknowledge the pertinent comment from Klocker et al. about the sex of rearing in individuals with perineoscrotal hypospadias and cryptorchidism. The adult phenotype of these individuals was not fully compatible with a traditional male gender role. They advocate a female sex of rearing, a recommendation we would heartily support. The promise of rapid molecular studies, as demonstrated in their study, may greatly facilitate our task in this regard. References 1. Plouffe L Jr: Androgen receptor locus on the human X chromosome: regional localization to the Xql1-12 and description of a DNA polymorphism (literature review) Adolesc Pediatr Gynecol 1989; 2: 195 2. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM: Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science 1988; 240:327 3. Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FS, Migeon CJ: Deletion of the steroidbinding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome. Proc Natl Acad Sci USA 1988; 85: 8151 4. Sai T, Seino S, Chang C, et al: An exonic point mutation of the androgen receptor gene in a family with complete androgen receptor insensitivity. Am J Hum Genet 1990; 46:1095 5. Marcelli M, Zoppi S, Grino PB, Griffin JE, Wilson JD, McPhaul MJ: A mutation in the DNA-binding domain of the androgen receptor gene causes complete testicular feminization in a patient with receptor-positive androgen resistance. J Clin Invest 1991; 87:1123
Literature Reviews
Pediatric Endocrinology Bone Mineralization during Female Adolescence Reviewed by: Frank B. Diamond, Jr., M.D. and Allen W. Root, M.D., Departments of Pediatrics (FBD, A WRY, Biochemistry and Molecular Biology (A WRY, University of South Florida College of Medicine, Tampa, and All Children's Hospital, St. Petersburg, FL Adolescence is a critical period for skeletal mineralization as more than half of adult bone mass is formed during puberty. Failure to accumulate adequate bone mineral reserves during adolescence may predispose to later osteopenia and its complications. Genetic factors, sex, skeletal and body size, race, hormonal milieu, diet, and physical exertion determine bone mass.! Thus, daughters of women with osteoporotic fractures of the hip or spine have lower bone mass than do daughters of normally mineralized women; there is greater correlation of bone mass between young adult monozygotic twins than between same sex dizygotic twins. Although bone mineral density (BMD) (g of calciumlm2) is similar, bone mineral content (BMC) (g of calcium) of adult males is greater than that of adult females, primarily due to the larger size of the male skeleton. Adult blacks have greater BMD and BMC than do white subjects. Estrogens, androgens, progestins, and resistance exercise increase bone mass. In eumenorrheic athletes, BMD may be greater than that of control subjects. 2 Inactivity, particularly immobilization, decreases bone mineralization. Dual energy x-ray absorptiometry (DEXA) is the preferred modality for assessment of bone mineralization because of its precision and low radiation dose. 3 Single and dual photon absorptiometry (SPA, DPA) and quantitative computed tomography (QCT) are less precise methods for measuring BMD and deliver higher doses of radiation. Furthermore, SPA measures BMD only in cortical compact bone, whereas DEXA, DPA, and QCT quantitate BMD in trabecular or spongy bone where bone mineral turnover is 8-fold more rapid than in compact bone, and thus more reflective of an alteration in bone synthesis or resorption. BMD and BMC of the vertebrae and long bones increase substantially during adolescence in both sexes (Fig. 1). The major increase in BMD and BMC of lumbar spine and femoral neck occurs earlier in girls (11-15 years) than in boys (13-17 years), and peak bone mass (maximal BMC) is achieved between ages 14 and 18 in girls and 17 and 18 in boys.4 Lumbar vertebral BMD of girls exceeds that of boys between 12 and 15 years of age, but BMD and BMC of the femoral neck and shaft are greater
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in adolescent boys than in girls. Approximately half of the pubertal increase in lumbar spine mineralization is due to enlargement of vertebral dimensions rather than to increased density per unit volume. s Early in adolescence the lumbar vertebral BMD of black and white girls is similar, but during late puberty black girls accrue bone mineral more rapidly than do white girls. 6 During adolescence, vertebral BMD correlates with chronologic age, height, weight, body surface area, skeletal maturation, and pubertal stage or "estrogen score," all factors of which are themselves intimately co-related. 4.7.8 The BMD of slim, low1.2
LUMBAR SPINE (L2·L4) BMD
1.1 1.0
0.7
10
11
12
13 14 15 AGE (years)
16
17
18
1.1
1.0
;
0.8
+1
1:
~ 0.8
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0.6 +--~~-~-~----_ 9 10 11 12 13 14 15 16 17 18 AGE (years)
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FEMORAL SHAFT BMD
2.0 1.8
~
+1
1:
~
1.6 1.4
1.2 1.0 +--~~---~----_ 9 10 11 12 13 14 15 16 17 18 AGE (years)
Fig. 1. Vertebral and femoral bone mineral density in female and male children and adolescents. (Reproduced from Bonjour et al: Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991; 73:555, copyright The Endocrine Society, with permission.)
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weight adolescents is less than that of heavier, stockier peers. In girls with anorexia nervosa, BMD is low. 9 Spinal and radial BMDs in 13 to 20-year-old females are related to an "estrogen score" (age of menarche, regularity of menses, Tanner breast stage, and estradiol level) that also reflects body size.? In mature 18 to 20-year-old females, BMDs are least in those with the lowest estrogen scores.? Amenorrheic subjects 15-21 years of age have lower lumbar spine BMDs than do eumenorrheic females of similar age, a difference primarily attributable to the lower weight of the amenorrheic subjects in this report. 10 In young women with amenorrhea and hypoestrogenism due to weight loss or excessive physical training, BMD is IOW. Il ,12 Delay in menarche and lengthy periods of amenorrhea predispose young ballet dancers to stress fractures. 12 Even in adult males with delayed adolescence, vertebral BMD is less than that of control subjects maturing at an earlier age. 13 Adequate dietary calcium is necessary for normal bone mineralization. The daily recommended dietary intake of elemental calcium for girls 11-18 years is 1,200-1,500 mg, 1,14 but most girls consume far less. 15 In a placebo, controlled twin study, supplementing dietary calcium intake (1,000 mg daily over 3 years) substantially increased BMD in prepubertal but not in pubertal subjects. 16 In adult females, vertebral and long bone mass correlate with calcium intake, but in children this relationship may be more variable. Il ,15 Smoking, excessive alcohol ingestion, illness, and lactation may diminish mineralization during adolescence. In conclusion, adolescence is a critical period for mineral acquisition which may have long-term effects on skeletal health. In young women, normal bone mineralization results from the interplay of genetics, diet, exercise, environmental factors, and hormonal milieu. References 1. Chesnut CH III: Theoretical overview: bone development, peak bone mass, bone loss and fracture risk. Am J Med 1991; 91(suppl 5B):25
2. Drinkwater BL, Nilson K, Chesnut CH III, et al: Bone mineral content of amenorrheic and eumenorrheic athletes. N Engl J Med 1984; 311:277 3. Johnston CC, Slemenda CW, Melton LJ: Clinical use of bone densitometry. N Engl J Med 1991; 324:1105 4. Bonjour J-P, Theintz G, Buchs B, et al: Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991; 73:555 5. Katzman DK, Bachrach LK, Carter DR, Marcus R: Clinical and anthropometric correlates of bone mineral acquisition in healthy adolescent girls. J Clin Endocrinol Metab 1991; 73:1332 6. Gilsanz V, Roe TF, Mora S, et al: Changes in vertebral bone density in black girls and white girls during childhood and puberty. N Engl J Med 1991; 325: 1597 7. Dhuper S, Warren MP, Brooks-Gunn J, Fox R: Effects of hormonal status on bone density in adolescent girls. J Clin Endocrinol Metab 1990; 71: 1083 8. Glastre C, Braillon P, David L, et al: Measurement of bone mineral content of the lumbar spine by dual energy x-ray absorptiometry in normal children: correlation with growth parameters. J Clin Endocrinol Metab 1990; 70: 1330 9. Bachrach LK, Guido D, Katzman D, et al: Decreased bone density in adolescent girls with anorexia nervosa. Pediatrics 1990; 86:440 10. White CM, Hergenroeder AC, Klish WJ: Bone mineral density in 15-21-year-old eumenorrheic and amenorrheic subjects. Am J Dis Child 1992; 146:31 11. Ott SM: Attainment of peak bone mass. J Clin Endocrinol Metab 1990; 71:1082A 12. Warren MP, Brooks-Gunn J, Hamilton LH, et al: Scoliosis and fractures in young ballet dancers. N Engl J Med 1986; 314:1348 13. Finkelstein JS, Neer RM, Biller BMK, et al: Osteopenia in men with a history of delayed puberty. N Engl J Med 1992; 326:600 14. Heaney RP: Effect of calcium on skeletal development, bone loss, and risk of fractures. Am J Med 1991; 91(suppl 5B):23S 15. Chan GM: Dietary calcium and bone mineral status of children and adolescents. Am J Dis Child 1991; 145:631 16. Johnson CC, Miller JZ, Slemenda SW, et al: Calcium supplementation and increases in bone mineral density in children. N Engl J Med 1992; 327:82
Adolescent and Pediatric Gynecology © 1993 Springer-Verlag New York Inc.
Literature Reviews Reproductive Endocrinology The Use of Antiandrogens in the Treatment of Benign Hirsutism in the Adolescent Reviewed by: Carol J. Levi, M.D. and Marvin L. Swanson, M.D., Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Sinai Hospital of Baltimore, Baltimore, MD. Antiandrogens are substances that prevent androgens from expressing their activity at target sites. These substances block androgenic activity in several ways: they modulate enzymatic activity, competitively bind to androgen receptors or testosterone-binding globulins, and/or increase aromatase activity.1,2 Interest in these compounds has grown recently regarding their use in hyperandrogenesis syndromes in females (e.g., hirsutism, polycystic ovarian disease (PCO), and acne). They have demonstrated promise in treating benign hirsutism. In a prior issue, we have reviewed the current data on oral contraceptive (OC) therapy for hirsutism in adolescents. This article will continue to examine therapies for benign hirsutism in adolescents, specifically focusing on antiandrogen therapies, and the new data emerging on this topic. Adolescents who present with benign hirsutism are a unique group in that they have specific concerns regarding self-image. OCs were examined in our previous review as an excellent, although not necessarily the only, treatment for benign adolescent hirsutism. OCs generally represent a good first line treatment for mild-to-moderate hirsutism in adolescents with the added benefit of providing contraception. They are packaged easily for the relatively noncompliant adolescent and have relatively few side effects. However, OCs are not always adequate treatment for the more severely affected benign hirsute patients, and other therapies may be needed. Antiandrogens, with or without OCs, may provide a more effective treatment in the adolescent with severe hirsutism. Five currently studied antiandrogens will be reviewed in this article: cyproterone acetate (CA), spironolactone (SP), flutamide (FL), cimetidine (CI), and ketoconazole (KC). CA and SP retain the steroid backbone common to estrogen and androgens, whereas FL, CI, and KC are nonsteroidal compounds. It should be remembered
that no medications are currently specifically approved by the FDA for the treatment of hirsutism. CA was originally developed as a progestin. It was found to have antiandrogenic activity because it caused feminization of male rat fetuses. It is a steroid antiandrogen that inhibits the formation of the nuclear androgen-receptor complex, and thus is primarily a competitive inhibitor of androgens. By decreasing levels of sex hormone binding globulin (SHBG), free serum testosterone (T) increases, which in turn increases its metabolic clearance rate. 3 In addition to being a potent progestin and an antiandrogen, it is also a weak glucocorticoid. CA is commercially available only in Canada and Europe. It is supplied in SO-mg doses or in combination with SO mcg or 3S mcg ethinyl estradiol as an OC in a 2-mg dose. The higher doses of CA are generally used to treat hirsutism. Because of CA's long half-life, typically 2S-100 mg can be given in the first 10--11 days of a birth control pill cycle. Side-effects of high-dose CA are similar to those of OCs in general, except that there seems to be more breast tenderness, amenorrhea, and weight gain with high-dose CA. As a treatment for hirsutism, it has been shown to significantly decrease hirsutism with single i.m. doses of 300 mg q month x 6 months which minimizes the side-effects of breast tenderness, amenorrhea, and weight gain. 3 ,4 Topical application of CA is ineffective. 2 Its use in a 2.0-mg dose in combination with ethinyl estradiol has been shown over a period of 3--6 months to significantly increase SHBG and decrease dehydroepiandrosterone sulfate (DHEA-S).4 Another study did not find a decrease in DHEA-S, suggesting that adrenal suppressive effects were unlikely. 3 The effect on CA in SHBG seems to be dose related, with higher doses resulting in decreased SHBG and the 2-mg dose resulting in increased SHBG. In any event, most of the patients noticed improvement in hirsutism between the eighth and twelfth cycle of treatment. Although it apparently will do well for the adolescent hirsute population, it awaits FDA approval for treatment in the U.S. and is currently not available. Spironolactone has been studied frequently in adults,5-8 but in relatively few adolescents. It has a steroid backbone structure, and acts as an aldosterone antagonist with antiandrogenic effects. AI-
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Literature Reviews
though the precise mechanism of antiandrogen action of SP is beyond the scope of this article, in general, SP decreases serum T, progesterone, and follicle-stimulating hormone (FSH) while increasing estradiol. 7 It inhibits the production of T by decreasing the microsomal cytochrome P450dependent enzymes. I Additionally, SP competes for androgen receptor binding sites, thus decreasing serum T levels. Three studies 6,8,9 found that serum androgens were not helpful in predicting the clinical response with SP in hirsute patients, suggesting that peripheral androgen metabolism is the major determinant of hirsutism in women. SP is equally effective in the treatment of idiopathic hirsutism and hirsutism associated with PCO. 8 Long-term therapy is important because the duration of therapy is directly related to clinical response. When it is administered at 100-200 mg/day (in two divided doses), facial hair became less coarse and growth occurred at a decreased rate. 7,10 It is important to realize that SP produces diuresis, decreases blood pressure, increases insulin levels,l1 as well as predisposes to menstrual irregularities. The menstrual irregularities result from the drug's effect on enzyme activity in the steroid pathways, as well as actions at the androgen and estrogen receptors. Adolescents may not tolerate these side effects and compliance may become an issue. Cyclic administration of SP may minimize side effects without decreasing efficacy, although this remains controversiaC,12 Prior characteristics of the menstrual cycle largely influence the incidence of menstrual irregularities. 13 OCs used in combination with SP will regulate potentially irregular cycles, and avoid this side effect. Although not considered to be a carcinogen,14 SP is a potential teratogen. Because it can prevent normal masculinization of a male fetus, its use in the adolescent population should probably be restricted to a combination with OCs to prevent pregnancy. Wild et al. II pointed out that the metabolic consequences of SP/OC treatment should be considered with respect to triglyceride and insulin levels and contraception. OCs tend to increase triglyceride levels whereas SP tends to increase insulin resistance. Insulin resistance appears to be at the root of a syndrome that predisposes to a cluster of metabolic disorders including atherosclerotic cardiovascular disease. 15 When OC/SP are used in combination, factors for cardiovascular disease increase. SP combined with an OC has been shown to improve hirsutism in women with irregular menses and other clinical features of PCO. This improvement was greater than when SP was used alone.23 Interestingly, SP has been tried as a topical therapy with fewer side effects. 12 Its use for acne was ef-
fective; however, its failure in the treatment of hirsutism attests to the importance of systemic levels of the drug which are not achieved topically. Flutamide (FL) is an anti androgen without the steroid backbone, therefore, it carries no glucocorticoid, progestational, androgenic, or estrogenic activities. 16 It has the ability to competitively block androgen action at the target tissue androgen receptor sites without decreasing the serum T level. Two studies evaluated the use of FL in hirsutism. In one study,16 it was administered in a dose of 250 mg b.i.d. in combination with ethinyl estradiol 35 mcg and norethindrone 1 mg with "good results," even in patients who had failed SP treatment. Adequate response required 6-12 months of combination therapy. The most common side effects included dry skin (70%), hot flushes (27%), and increased appetite (25%) although weight gain was not reported. None of the study subjects dropped out of this clinical trial because of side effects. In a more recently published clinical trial,17 FL was given alone in a dose of 250 mg Li.d. to nine hirsute women for 3 months. The FerrimanGallwey scores of assessment of body hair growth in women were significantly improved. Side effects included a high incidence of oligomenorrhea, and a low incidence of dry skin. No other serious side effects were reported. Interestingly, hormonal analysis of the study subjects showed no changes in serum androgens, or in the measured or calculated T fractions, suggesting that FL acts only at the androgen receptor level. This is different than the two other antiandrogens, CA and SP, both of which change the serum androgen levels as previously discussed. This medication may have promise in adolescent hirsutism. More studies will be forthcoming. Cimetidine, a histamine receptor type 2 blocker, has been used as therapy for hirsutism. It blocks the action of androgens at the hair follicle by inhibiting the binding of dihydrotestosterone to androgen receptors. However, earlier reports of success have been dampened by more recent reports of it failures,18.19 and appears not to be effective clinically. Ketoconazole, an antimycotic agent, has also been shown to reduce serum levels ofT, DHEA-S, and androstenedione,zo However, clinical efficacy has been mixed 21 .22 and side effects of mastodynia, gastrointestinal upset, and somnolence may limit its use. Clinical Correlation. In summary, the treatment of hirsutism recalcitrant to oral contraceptives should begin with SP. SP use should be monitored with respect to its side effects, and concomitant contraception is required with its use. Although CA appears to be very effective against
Literature Reviews
hirsutism, it is not yet commercially available in the U.S. Flutamide, although a promising antiandrogen, requires further long-term evaluation before its use is widespread. The hirsute adolescent may require prolonged therapy to successfully decrease unwanted hair growth. A balance between aggressive treatment and multiple side effects will continue to be sought for optimum care of this unique patient population. References 1. Sciarra F, Toscano V, Concolino G, Di Silverio F: Antiandrogens: clinical applications. J Steroid Molec Bioi 1990; 37:349 2. Neumann F, Topert M: Pharmacology of antiandrogens. J Steroid Biochem 1986; 25:885 3. Marcondes JAM, Wajchenberg, BL, Abujamra AC, et al: Monthly cyproterone acetate in the treatment of hirsute women: clinical and laboratory effects. Fertil Steril 1990; 53:40. 4. Prelevic GM, Wurzburger MI, Balint-Peric L, et al: Effects of a low-dose estrogen-antiandrogen combination (Diane-35) on clinical signs of androgenization, hormone profile and ovarian size in patients with polycystic ovary syndrome. Gynecol Endocrinol 1989; 3:269 5. Vetr M: Hirsutism-a low dose spironolactone therapy. Acta Univ Palacki Olomuc Fac Med 1989; 122 6. Spandri P, Gangemi M, Nardelli GB, et al: Testosterone, 17KS, 1713 E 2 , FSH-LH variations and hirsutism modifications during spironolactone therapy. Clin Exp Obst Gyn 1984; 1-2:49 7. Shapiro G, Evron S: A novel use of spironolactone: treatment of hirsutism. J Clin Endocrinol Metab 1980; 51:429 8. Evans DJ, Burke CW: Spironolactone in the treatment of idiopathic hirsutism and the polycystic ovary syndrome. J Royal Soc Med 1986; 79:451 9. Carmina E, Lobo RA: Peripheral androgen blockade versus glandular androgen suppression in the treatment of hirsutism. Obstet Gynecol 1991; 78:845 10. Barth JH, Cherry CA, Wojnarowska F, et al: Spironolactone in an effective and well-tolerated systemic antiandrogen therapy for hirsute women. J Clin Endocrinol Metab 1989; 68-5:966 11. Wild RA, Demers LM, Applebaum-Bowden D, et al: Hirsutism: metabolic effects of two commonly used oral contraceptives and spironolactone. Contraception 1991; 44: 113 12. Messina M, Manier C, Musso MC, et al: Oral and topical spironolactone therapies in skin androgenization. Panminerva Med 1990; 32:49 13. Helfer EL, Miller JL, Rose LI: Side effects of spironolactone therapy in the hirsute woman. J Clin Endocrinol Metab 1988; 66-1 :208 14. Lumb G, Newberne P, Rust JH, et al: 1978 Effect in animals of chronic administration of spironolactone: a review. In: Aldosterone antagonists in Clinical
15.
16. 17. 18.
19. 20.
21.
22. 23.
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Medicine. Edited by Addison GM, Asmussen NW, Corvol P, Kloppenborg PWC, Norman M, Schroder R, Robertson nc. Amsterdam, Oxford, Excerpta Medica, p 132 DeFronzo RA, Ferrannini E: Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991; 14: 173 Cusan L, Dupont A, Belanger A, et al: Treatment of hirsutism with the pure antiandrogen flutamide. J Am Acad Derm 1990; 23:462 Marcondes JAM, Minnani SL, Luthold WW, et al: Treatment of hirsutism in women with flutamide. Fertil Steril 1992; 57:543 Lissak A, Sorokin Y, Calderon I, et al: Treatment of hirsutism with cimetidine: a prospective randomized controlled trial. Fertil Steril 1989; 51 :247 Golditch 1M, Price VH: Treatment of hirsutism with cimetidine. Obstet Gynecol 1990; 75:911 DePedrini P, Tommaselli A, Spano G, et al: Clinical and hormonal effects of ketoconazole on hirsutism in women. Int J Tissue Reac 1988; 3:193 Conget n, Halperin I, Ferrer J, et al: Evaluation of clinical and hormonal effects of hirsute women treated with ketoconazole. J Endocrinol Invest 1990; 13:867 So nino N, Scaroni C, Biason A, et al: Low-dose ketoconazole treatment in hirsute women. J Endocrinol Invest 1990; 13:35 Rittmaster RS: Evaluation and treatment of hirsutism. Fertil Reprod CI N Am 1991; 2:511
Molecular Biology Evidence for a Partial Deletion in the Androgen Receptor Gene in a Phenotypic Male with Azoospermia. Akin JW, Behzadian A, Tho SPT, McDonough PG. Am J Obstet Gynecol 1991;165:1891 and Point Mutation in the DNA Binding Domain of the Androgen Receptor in Two Families with Reifenstein Syndrome. Klocker H, Kaspar F, Eberle J, Uberreiter S, Radmayr C, Bartsch G. Am J Hum Genet 1992;50: 1318. Reviewed by: Leo Plouffe, Jr., M.D., C.M., Section of Reproductive Endocrinology, Genetics and Infertility, Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA. We have previously reviewed the initial report about the localization of the androgen receptor (AR) gene.! The locus, at the Xql1-12 region, has been the subject of intense research since the report by Lubahn et al. 2 Many investigators have described mutations in the AR gene associated with complete androgen receptor insensitivity. 3-5 The two current reports highlight a similar type of gene
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Literature Reviews
mutation associated with partial androgen receptor defects. Several authors over the years have proposed that azoospermia and oligospermia may result from defects in the androgen receptor. Picking up on this idea, Akin et al. studied seven otherwise normal males who presented with azoospermia. One of these seven men was found to have a partial gene deletion in exon 4, a region responsible for DNA binding. This was visible through both polymerase chain reaction (PCR) and Southern blot analysis. Unfortunately, these authors did not present family studies to confirm the association of this mutation with azoospermia. Ideally, one would like to find a similar mutation in the patient's mother and other affected males within the family. Following these studies, the exact nature of the mutation should be determined. Nonetheless, this constitutes one of the first reports of a mutation in the AR gene associated with a very limited phenotypic manifestation. The report by Klocker et al. is impressive for both the breadth of the patients studied and the thoroughness of their methodology. These authors present reports on two families. However, they studied a total of 41 inter-sex patients. Of this group, they identified five study subjects who presented with perioneoscrotal hypospadias and undescended testes, a phenotype compatible with Refenstein syndrome. Three siblings from one family were studied, along with their mother, sister, one aunt, and one uncle. Two affected males from another family were studied along with blood from their mother. Genital skin fibroblasts were obtained from all patients and were studied for specific binding of dihydrotestosterone, 5-a reductase activity. Both were within normal range. The defect they identified is in exon 3, again part of the DNA binding domain of the androgen receptor. The mutation consists of a guanine to adenine substitution at nucleotide 2314. This produces the substitution of threonine for alanine in a zinc finger region of the AR. As a result of this mutation, androgen binding to its receptor is not affected but the activity of the androgen-receptor complex is diminished. A male phenotype does result but undermasculinization is evident. The family information confirms the X-linked nature of this disorder, with the demonstration of the same mutation in the mother and sister of one family. For the molecular biologists among us, the technique utilized to rapidly screen for the presence of the mutation is of particular interest. It involves both PCR and restriction-analysis method. This rapid methodology is particularly suited for clinical applications and is quickly gaining popularity.
Clinical Correlation. The two papers reviewed here point to the varied phenotypes that can be associated with defects in the AR gene. This is now a good model for a genetic disorder in reproductive medicine. One should consider such a defect whether dealing with a child with ambiguous genitalia, a male teenager with pubertal delay, a female with the phenotype of complete androgen insensitivity, or a male with azoospermia. Could it also be that males with cryptorchidism or oligospermia also carry minor mutations in the AR gene? Only time will tell. We can also speculate that some mutations may result in overexpression of androgen action, as has recently been described for McCune-Albright disease and cGMP. In this context, we may gain new insights into the complex problems of hirsutism and masculinization in the female. As a final note, we would like to acknowledge the pertinent comment from Klocker et al. about the sex of rearing in individuals with perineoscrotal hypospadias and cryptorchidism. The adult phenotype of these individuals was not fully compatible with a traditional male gender role. They advocate a female sex of rearing, a recommendation we would heartily support. The promise of rapid molecular studies, as demonstrated in their study, may greatly facilitate our task in this regard. References 1. Plouffe L Jr: Androgen receptor locus on the human X chromosome: regional localization to the Xql1-12 and description of a DNA polymorphism (literature review) Adolesc Pediatr Gynecol 1989; 2: 195 2. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM: Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science 1988; 240:327 3. Brown TR, Lubahn DB, Wilson EM, Joseph DR, French FS, Migeon CJ: Deletion of the steroidbinding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome. Proc Natl Acad Sci USA 1988; 85: 8151 4. Sai T, Seino S, Chang C, et al: An exonic point mutation of the androgen receptor gene in a family with complete androgen receptor insensitivity. Am J Hum Genet 1990; 46:1095 5. Marcelli M, Zoppi S, Grino PB, Griffin JE, Wilson JD, McPhaul MJ: A mutation in the DNA-binding domain of the androgen receptor gene causes complete testicular feminization in a patient with receptor-positive androgen resistance. J Clin Invest 1991; 87:1123
Literature Reviews
Pediatric Endocrinology Bone Mineralization during Female Adolescence Reviewed by: Frank B. Diamond, Jr., M.D. and Allen W. Root, M.D., Departments of Pediatrics (FBD, A WRY, Biochemistry and Molecular Biology (A WRY, University of South Florida College of Medicine, Tampa, and All Children's Hospital, St. Petersburg, FL Adolescence is a critical period for skeletal mineralization as more than half of adult bone mass is formed during puberty. Failure to accumulate adequate bone mineral reserves during adolescence may predispose to later osteopenia and its complications. Genetic factors, sex, skeletal and body size, race, hormonal milieu, diet, and physical exertion determine bone mass.! Thus, daughters of women with osteoporotic fractures of the hip or spine have lower bone mass than do daughters of normally mineralized women; there is greater correlation of bone mass between young adult monozygotic twins than between same sex dizygotic twins. Although bone mineral density (BMD) (g of calciumlm2) is similar, bone mineral content (BMC) (g of calcium) of adult males is greater than that of adult females, primarily due to the larger size of the male skeleton. Adult blacks have greater BMD and BMC than do white subjects. Estrogens, androgens, progestins, and resistance exercise increase bone mass. In eumenorrheic athletes, BMD may be greater than that of control subjects. 2 Inactivity, particularly immobilization, decreases bone mineralization. Dual energy x-ray absorptiometry (DEXA) is the preferred modality for assessment of bone mineralization because of its precision and low radiation dose. 3 Single and dual photon absorptiometry (SPA, DPA) and quantitative computed tomography (QCT) are less precise methods for measuring BMD and deliver higher doses of radiation. Furthermore, SPA measures BMD only in cortical compact bone, whereas DEXA, DPA, and QCT quantitate BMD in trabecular or spongy bone where bone mineral turnover is 8-fold more rapid than in compact bone, and thus more reflective of an alteration in bone synthesis or resorption. BMD and BMC of the vertebrae and long bones increase substantially during adolescence in both sexes (Fig. 1). The major increase in BMD and BMC of lumbar spine and femoral neck occurs earlier in girls (11-15 years) than in boys (13-17 years), and peak bone mass (maximal BMC) is achieved between ages 14 and 18 in girls and 17 and 18 in boys.4 Lumbar vertebral BMD of girls exceeds that of boys between 12 and 15 years of age, but BMD and BMC of the femoral neck and shaft are greater
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in adolescent boys than in girls. Approximately half of the pubertal increase in lumbar spine mineralization is due to enlargement of vertebral dimensions rather than to increased density per unit volume. s Early in adolescence the lumbar vertebral BMD of black and white girls is similar, but during late puberty black girls accrue bone mineral more rapidly than do white girls. 6 During adolescence, vertebral BMD correlates with chronologic age, height, weight, body surface area, skeletal maturation, and pubertal stage or "estrogen score," all factors of which are themselves intimately co-related. 4.7.8 The BMD of slim, low1.2
LUMBAR SPINE (L2·L4) BMD
1.1 1.0
0.7
10
11
12
13 14 15 AGE (years)
16
17
18
1.1
1.0
;
0.8
+1
1:
~ 0.8
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0.6 +--~~-~-~----_ 9 10 11 12 13 14 15 16 17 18 AGE (years)
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FEMORAL SHAFT BMD
2.0 1.8
~
+1
1:
~
1.6 1.4
1.2 1.0 +--~~---~----_ 9 10 11 12 13 14 15 16 17 18 AGE (years)
Fig. 1. Vertebral and femoral bone mineral density in female and male children and adolescents. (Reproduced from Bonjour et al: Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991; 73:555, copyright The Endocrine Society, with permission.)
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weight adolescents is less than that of heavier, stockier peers. In girls with anorexia nervosa, BMD is low. 9 Spinal and radial BMDs in 13 to 20-year-old females are related to an "estrogen score" (age of menarche, regularity of menses, Tanner breast stage, and estradiol level) that also reflects body size.? In mature 18 to 20-year-old females, BMDs are least in those with the lowest estrogen scores.? Amenorrheic subjects 15-21 years of age have lower lumbar spine BMDs than do eumenorrheic females of similar age, a difference primarily attributable to the lower weight of the amenorrheic subjects in this report. 10 In young women with amenorrhea and hypoestrogenism due to weight loss or excessive physical training, BMD is IOW. Il ,12 Delay in menarche and lengthy periods of amenorrhea predispose young ballet dancers to stress fractures. 12 Even in adult males with delayed adolescence, vertebral BMD is less than that of control subjects maturing at an earlier age. 13 Adequate dietary calcium is necessary for normal bone mineralization. The daily recommended dietary intake of elemental calcium for girls 11-18 years is 1,200-1,500 mg, 1,14 but most girls consume far less. 15 In a placebo, controlled twin study, supplementing dietary calcium intake (1,000 mg daily over 3 years) substantially increased BMD in prepubertal but not in pubertal subjects. 16 In adult females, vertebral and long bone mass correlate with calcium intake, but in children this relationship may be more variable. Il ,15 Smoking, excessive alcohol ingestion, illness, and lactation may diminish mineralization during adolescence. In conclusion, adolescence is a critical period for mineral acquisition which may have long-term effects on skeletal health. In young women, normal bone mineralization results from the interplay of genetics, diet, exercise, environmental factors, and hormonal milieu. References 1. Chesnut CH III: Theoretical overview: bone development, peak bone mass, bone loss and fracture risk. Am J Med 1991; 91(suppl 5B):25
2. Drinkwater BL, Nilson K, Chesnut CH III, et al: Bone mineral content of amenorrheic and eumenorrheic athletes. N Engl J Med 1984; 311:277 3. Johnston CC, Slemenda CW, Melton LJ: Clinical use of bone densitometry. N Engl J Med 1991; 324:1105 4. Bonjour J-P, Theintz G, Buchs B, et al: Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991; 73:555 5. Katzman DK, Bachrach LK, Carter DR, Marcus R: Clinical and anthropometric correlates of bone mineral acquisition in healthy adolescent girls. J Clin Endocrinol Metab 1991; 73:1332 6. Gilsanz V, Roe TF, Mora S, et al: Changes in vertebral bone density in black girls and white girls during childhood and puberty. N Engl J Med 1991; 325: 1597 7. Dhuper S, Warren MP, Brooks-Gunn J, Fox R: Effects of hormonal status on bone density in adolescent girls. J Clin Endocrinol Metab 1990; 71: 1083 8. Glastre C, Braillon P, David L, et al: Measurement of bone mineral content of the lumbar spine by dual energy x-ray absorptiometry in normal children: correlation with growth parameters. J Clin Endocrinol Metab 1990; 70: 1330 9. Bachrach LK, Guido D, Katzman D, et al: Decreased bone density in adolescent girls with anorexia nervosa. Pediatrics 1990; 86:440 10. White CM, Hergenroeder AC, Klish WJ: Bone mineral density in 15-21-year-old eumenorrheic and amenorrheic subjects. Am J Dis Child 1992; 146:31 11. Ott SM: Attainment of peak bone mass. J Clin Endocrinol Metab 1990; 71:1082A 12. Warren MP, Brooks-Gunn J, Hamilton LH, et al: Scoliosis and fractures in young ballet dancers. N Engl J Med 1986; 314:1348 13. Finkelstein JS, Neer RM, Biller BMK, et al: Osteopenia in men with a history of delayed puberty. N Engl J Med 1992; 326:600 14. Heaney RP: Effect of calcium on skeletal development, bone loss, and risk of fractures. Am J Med 1991; 91(suppl 5B):23S 15. Chan GM: Dietary calcium and bone mineral status of children and adolescents. Am J Dis Child 1991; 145:631 16. Johnson CC, Miller JZ, Slemenda SW, et al: Calcium supplementation and increases in bone mineral density in children. N Engl J Med 1992; 327:82