J. Endocrino!. Invest. 14: 153-170, 1991
Hirsutism: pilosebaceous unit dysregulation. Role of peripheral and glandular factors V. Toscano Istituto di V Clinica Medica, Policlinico Umberto I, Universita La Sapienza, 00161 Rome, Italy
patient. A degree of 5 or more may be considered excessive hair growth , if hair are distributed in more than 2 areas (Fig . 1). Both Ferriman and Gallwey's and Lorenzo's scales do not take into account the fact that abnormal amounts of hair growth may be confined to only one or two areas without raising the total hirsutism score , In these cases , in fact, even if the score of 5 was not reached because hirsutism was confined in only one or two areas , the psychological distress has to be taken into account in the final evaluation for defining the patients affected by excessive hair growth. Hirsutism must be distinguished from hypertrichosis, in which the increase in hair growth is limited to the sites normally-covered by hair in women , and from virilism in which hirsutism is only one clinical manifestation of complete defeminization (deeping in the voice, increased muscle mass, clitoromegaly etc .) The pathogenesis of hirsutism is not clear: hirsutism may develop in presence of normal androgen production and may be absent in some case of hyperandrogenism . This review outlines the central role of pilosebaceous unit disregulation in hirsutism induction, considering the possible interference of other factors which can affect the androgens availability at skin level.
Hirsutism is defined as excessive terminal hair on body skin areas where such growth is considered male secondary sexual characteristic . A pioneer study on hair growth assessment dates to 1922 (1) , but the first important study (Ferriman and Gallwey , (2) suggesting a method for the semiquantitative assessment of body hair appears in 1961 . These authors consider hair distribution in 11 areas grading the density of terminal hairs from 0 (absence of terminal hair) to 4 (frankly virile) . They defined "hormonal score " as the value obtained adding the gradings obtained from 9 of the 11 sites (excluding forearm and legs) . McKnight (3) described the amount of body hair in normal British women and found that 18% had chest hair, 26% had facial hair, 35% had abdominal hair and 70% had hair in the upper arms or legs , less than 5% had an hirsutism score of 8 or more using the scale of Ferriman and Gallwey. Lorenzo (4) in 1971 proposed a modification of the estimation procedure of Ferriman and Gallwey. Madanes and Novotny (5) proposed a method evaluating the percent of vellus hair in a miscroscopic observation of shaved hairs from one area of the body . This method seems complicated and probably more useful for the evaluation of treatment results than for assessment of hair growth . Analogous consideration could be drawn for the method proposed by Casey (6) and Cumming (7) . We use a modification of Lorenzo's scale taking into consideration hair distribution in 5 sites (chin, upper lip , chest, abdomen and thighs). The severity of hirsutism in each of these areas was graded from 1 (least severe) to 4 (most severe) , the sum of the scores representing the degree of hirsutism in each
PHYSIOLOGY OF PILOSEBACEOUS UNIT
Key-words: Hirsutism . acne. PCO , congenital adrenal hyperplasia.
The pilosebaceous units which consist of pilary and sebaceous components are formed between the second and fourth month of fetal life and begin to function in utero. This structure represents a target for hormonal action , androgen in particular. The degree of androgen sensitivity and the morphology of this structure are secondary to the localization . During fetal life the pilosebaceous unit sensitivity to androgen is probably set in accordance with genetic control (8) .
Correspondence: Dr. Vincenzo Toscano , Istituto di V Clinica Medica, Policlinico Umberto I, Universita La Sapienza 0016 1 Roma, Italy.
behave identically and their response to androgen
However the hair and the sebaceou s glands do not
Fig. 1 - Hair growth gradings. In each area hirsutism was graded from 1 (least severe) to 4 (most severe).
stimulation vary greatly among sites. On this basis we can consider non androgen-sensitive areas, including scalp and eyebrows and androgen-sensitive areas which include sebaceous areas and sexual hair areas, both ambosexual (lower pubic triangle or axilla) and masculin hairs (moustache and beard). Before puberty in androgen sensitive areas the hair is vellus (fine, pale and short hair) and sebaceous glands are small. At puberty, under androgen stimulation terminal hair (longer, thicker and darker than vellus) develops. Hair grows cyclically (9). The active growth period is called the anagen phase . The telogen phase is a resting phase. Between these two phases a transitional phase is described (catagen). Once a new hair starts to grow, the old telogen shaft is pushed out and the cycle begins again. The amount of hair growth in any area of the body depends on several factors: duration of anagen and percent of hairs in this phase ; linear growth rate; diameter of hairs and density of terminal hairs. (10-11). Androgens are the principal hormones responsible for hair growth: they trigger the matrix cells of the pilosebaceous unit to produce terminal hairs rather than vellus hairs, and increase the time spent in anagen. The growth of pubic and axillary hair require low levels of androgens (14) . The growth of hair on the scalp are androgen-independent and paradoxically the androgens are a prerequisite for manifestation of male pattern alopecia (15, 16). Facial hairs as the hairs in the trunk and extremities require the 5a reduced metabolite of testosterone (T), the dihydrotestosterone (OHT) to grow, but the density of the hairs correlated only with T. That explains the obvious sexual differences in hair growth between females and males . However, in considering variations between individuals, the extent of the peripheral response may be even more important than the level of circulating androgen. In other words , the capacity of the hair follicle to metabolize T is more important than the level of T available (12-13), outlining the central role of pilosebaceous unit. Some authors on the basis of in vitro experiments using cultured skin cells and evaluating the pattern of incorporation of 35S-cysteine or 35Smethionine and the formation of DHT from T conclude that the interaction of epithelial with mesenchymal cells are mandatory for the action of androgen on hair and other epithelial cell types and suggest that androgen may act at two or more sites in modulating hair growth (16-18). Estrogens stimulate pubic and axillary hair growth
and probably induce androgen receptors (19) or increase insulin-like growth factors (20). Insulin-like growth factors (IGF-I) probably synergize with androgen in hair growth. Zachmann and Prader (21) showed in GH-deficient children that pubic and axillary hair are subnormally responsive to testosterone. It is also known that pubertal amounts of testosterone elevate plasma levels of IGF·I above adult levels, via stimulation of GH secretion (20). Epidermal growth factor, which seems to be important in epidermal cell differentiation, may also playa role in anagen hair growth as it increases keratinocytes proliferation (22-23), probably mediating androgen action (24), which seems to be very important in promoting follicular keratinization (25). Sebaceous glands are holocrine; they occur throughout the human skin except for the palms and the soles and dorsum of the feet. The role of androgens in sebaceous secretion may be inferred by the fact that the sebum levels in the first week of life are as high as in an adult (26), when androgen levels are also high. The sebum production then declines until adrenarche,when the production begins to increase again (27). In men the sebum levels decline around the age of 80 and in women, who excrete as much sebum as men, the amount falls off after the age of 50 (28-29). From this data it can be inferred that sebum production is very sensitive to androgens and also very low levels of these are sufficient to stimulate it; higher levels of androgens sharply increase the amount of sebum. An important individual variability in the amount of sebum produced to a given amount of androgen, could explain the large difference observed in several patients showing similar androgen plasma level (27). Sebaceous cells showed all of the biochemical machinery necessary for androgen action: they can form DHT from T and dehydroepiandrosterone (DHA) and possess androgen receptors (30-33) . Estrogens depress sebaceous activity, in turn depressing gonadotropin secretion and switching off gonadal androgen production both in men and women. Glucocorticoids may exert their stimulatory effect on sebum production potentiating androgen action. Progesterone is ineffective at physiological doses. Thyroxine seems to increase sebum production (34). Hypophyseal hormones (ACTH, LH, TSH) probably exert their positive effect on sebum production increasing adrenal, gonadal and thyroidal hormones. The demonstration however that in the panhypopituitaric rat replacement therapy does not completely improve the sebum production leads one to postulate a direct effect. To explain this, var-
ious authors have attributed this direct effect to prolactin, melanocyte stimulating hormone, and Blipotropin (34-37). GH, as postulated for sexual hair growth, probably exert a synergistic effect with androgen in sebum production. This effect is probably mediated by IGF-I (20).
amount of T (about 20% of its daily production). Their production increases during adrenarche, reaching a peak during reproductive life and decline in old age. This secretory pattern completely different from cortisol lead to acceptance of the existence of a tropic hormone for adrenal androgen production different from ACTH (42-47). The graffian follicle, corpus luteum and stroma represent the areas in the ovary where androgen biosynthesis occurs. In the follicle and stroma 5ene pathway is favoured, while in the corpus luteum 4-ene pathway appears to be favorred (48). Androstenedione represents the androgen secreted in greater amount by the ovary. Testosterone is also secreted in very small amount, comparable to that secreted by the adrenals. DHA is also secreted in very small amount. Ovarian androgen production is regulated by gonadotropin stimulation, LH in particular. Since LH changes enormously during the menstrual cycle, the ovarian contribution to the daily production of androgen varies as well from the lowest in early follicular phase to the higher levels prior to, or at the time of, ovulation and then gradually falling during the luteal phase (49). In post-menopausal women secretion of androgens usually continues and probably represents the continued stimulation of stromal tissue by the elevated levels of LH (50). Ovarian androgen production seems to be regulated by several factors which stimulate or inhibit LH action. Among these insulin seems to playa very important role. Among the inhibitory factors prolactin and GnRH-like substances should also be mentioned (51).
PHYSIOLOGY OF ANDROGEN PRODUCTION
Androgens were described as substances "capable of stimulating male secondary sex characteristics" (38). Chemically, androgens are C-19 steroids with a flat junction between rings A and B and an oxygenated function on the 17th carbon. Between these molecules we can distinguish active androgens from inactive precursors or metabolites considering active molecules those capable, as dihydrotestosterone, of binding,with specific high affinity, to nuclear receptor interacting with chromatin and inducing new messanger RNA synthesis. Androgen precursors of active androgens, also defined as prehormones, could be unconjugated or conjugated. Similarly, metabolites or peripheral androgens could be unconjugated and conjugated. Ovary and adrenal gland account for a large part of production of androgen precursors in women. ANDROGEN BIOSYNTHESIS
Steroidogenic tissues may synthesize cholesterol de novo from acetate (39), but in men steroidogenic cells derive most of their cholesterol from plasma low density lipoprotein (LDL) (40). Tropic hormones stimulate LDL receptor and uptake of LDL cholesterol. Cholesterol storage as cholesterol esters in lipid droplets is controlled by the action of two opposing enzymes, cholesterol esterase and cholesterol ester synthetase. Tropic hormones, like ACTH or LH, stimulate the esterase and inhibit the synthetase, increasing the availability of free cholesterol for steroidogenetic steps. Free cholesterol is transported to the mitochondria, where it is converted to pregnenolone, which represents the first rate limiting and hormonally regulated step in the synthesis of all steroid hormones (41 ). From this first step the steroid biosynthesis follows two different pathways, the 4-ene and 5-ene, as shown in Figure 2.
The 5a reduction is the key step for androgen action at target tissue with formation of DHT, which represents the nuclear mediator of androgen action. 5a reductase is an NADPH-dependent, membrane-bound, noncytochrome P-450-dependent enzyme, apparently associated with nuclear membrane. Studies based on enzyme kinetics suggest the presence of two isoenzymes, the results of genetic studies in human 5a reductase deficiency indicate the presence of only one gene (41, 52). In women the major precursor of DHT is androstenedione, accounting for 60%. T account for the 15% of DHT and DHA and androstenediol for the remaining 25% (52). In extrasplanchnic sites, where DHT is almost completely sinthetized at least in normal women, it is further metabolized to 3a and 3B androstanediols and 3a androstanediol glucuronide before enter-
The principal androgens secreted by the adrenal glands in women are represented by DHA and its sulfate (DHA-S), androstenedione and small
~! Fig.2 - Biosynthetic patways of androgens and estrogens. Numbers indicate enzymes 1: 20,22 desmolase 2: 38 -ol-hydroxysteroid dehydrogenase 3: 17-hydroxylase 4: 17,20-lyase 5: 17-ketosteroid reductase 6: Sa reductase 7: aromatase
Androgen plasma carriers Albumin and sex hormone binding globulin (SHBG) enhance the solubility of androgens and facilitate their transport from steroidogenic sites to target cells. Albumin represents a non specific protein with large capacity, while SHBG represents the carrier with very high specificity and low capacity and highest affinity for DHT. It is not clear yet if the SHBG affects steroid entry into cells (56-58). Very recent data of Rosner and coworkers (59, 60) showed that SHBG, like cortisol binding globulin (CBG), has a membrane receptors in steroid target cells and this protein has two binding sites, one
ing the general circulation, so that circulating DHT may not reflect its peripheral formation (52-54) (Fig. 3A). 3a androstanediol and its glucuronide on the contrary seem to reflect better peripheral androgen formation (53), being also demonstrated that these metabolites derive exclusively from extrasplanchnic androgen metabolism. As far as 3a androstanediol glucuronide is concerned, it has been recently demonstrated by Rittmaster that the predominant circulating form is represented by the 17 conjugated, which also is the major isomer derived from DHT, both in men and women (55).
binds steroids and the other binds to a membrane receptor. The interaction of SHBG with this receptor induce an intracell.ular accumulation of cAMP, increasing the adenylate cyclase. This effect however is minimal if unliganded SHBG alone bound to the membrane receptor, the addiction of androgen , which bind its receptor on the protein, increases the cAMP accumulation into the cells. These studies showing that plasma steroid-binding protein could. be effectors for a second messenger system, however they still do not furnish data about the role of the binding protein in the mechanism for steroid entry into target cells (Fig. 3B).
Table 1 - Causes of hirsutism Peripheral Increased Sa reductase activity (primary or secondary)
Qualitative or quantitative defect of Sex-Hormone BindingProtein (primary or secondary) Glandular Adrenal Congenital Adrenal Hyperplasia (classic and non classic forms) : - 21-hydroxylase deficiency - 3-B-ol-dehydrogenase deficiency - 11-hydroxylase deficiency
Adrenal tumors Ovarian Polycystic ovary syndrome due to increased intraovarian production of androgens for: - genetic defect of intraovarian steroidogenesis - increased synthesis by insulin or other growth factors.
Ovarian tumors Exogenous medications Anabolic steroids Danazol Minoxidil Diamox Phenothiazine Birth control pills (androgenic progestins)
DHA DHAS A Plasma precursors
? " +
When presented with a patient complaining of excess hair growth , the physician has fo consider the possible causes by performing a correct anamnesis and an adequate exclusionary workup prior to starting the therapy. This requires an understanding of the etiology of hirsutism and the clinically available tests that may distinguish between them, Therefore it is important to know the pathogenetic mechanisms leading to the hirsutism. Table 1 lists possible causes of hirsutism.
T-SHBG SHBG B
Fig,3 - Cellular androgen action
T: testosterone OHA: dehydroepiandrosterone OHA-S: dehydroepiandrosterone sulfate FT: free testosterone OHT: dihydrotestosterone 3 Ad:3a androstanediol 3 AdG:3a androstanediol glucuronide T-SHBG.Sex Hormone Binding Globulin complexed with steroid SHBG:Sex Hormone Binding Globulin
As it was said, the key for androgen action is represented by 5a reduction of T to DHT at target tissue sites including hair follicles. Incubation of skin from hirsute women (61-64) demonstrated more active transformation of T to DHT compared to normal women. Mauvais-Jarvis and coworkers first demonstrated that 5a reductase is primitively increased in those hirsute patients in which androgen levels were normal (61-64). On the contrary no differences in the
A: classical view B. new view A: androstenedione
plasma proteins in vivo (78). These contradictory results about the possibility that FFA could interfere in the free androgen availability in vivo and in free fraction evaluation in vitro need to be clarified. There is direct evidence that SHBG and then bioactive androgens could be influenced by dietary lipids (86, 87), but the mechanism by which they can influence SHBG is not clear. A direct or indirect effect on hepatic SHBG synthesis has been hypothesized by Reed (86). It is also very well known that obese non hirsute patients show low levels of SHBG. An inverse relationship of serum SHBG concentrations with BMI is now well recognized and probably this effect may be mediated by insulin (88, 89). On the contrary a low calorie diet, which reduces body weight, increases SHBG levels with a parallel increase of IGF I binding protein, which is known to be insulin dependent (90). A diet including a lipid excess or obesity could therefore represent other important peripheral factors modulating bioactive androgen availability for target sites and then represent a critical factors for clinical manifestations of hyperandro§:)enism. The role of adipose tissue is complex. This tissue is able to actively transform T to DHT (91) and to androstanediols, but may serve to deactivate at least in part, bioactive androgens into inactive metabolites (92). On the other hand adipose tissue can convert preandrogens into T, contributing to the increased T production rate observed in obese patients (93).
binding capacity of genital skin fibroblasts were found between normal and hirsute women and men (65-68), so that increased androgen binding capacity cannot explain excess hair growth in hirsute women. Serafini and Lobo also found in the skin of idiopathic hirsute patients high levels of 5a reductase activity (69) and this increased conversion correlate well with the severity of hirsutism (70). It is however still unclear if this increased 5a reductase activity is the expression of a genetic disorder or if it expresses the results of abnormally set androgen receptors so that also very low levels of active androgens or normal levels of week androgens may increase the enzymatic activity. IGF I has been ~hown to be a potent inducer of 5a reductase activity in cultured scrotal rat fibroblasts (71). The induction of this enzyme by factors other than androgens open a new way for understanding the pathogenesis of those forms of hirsutism presenting normal plasma androgens (idiopathic hirsutism) (Fig. 3A, B). Idiopathic hirsutism is frequently associated with low levels of plasma SHBG, allowing a higher fraction of bioactive androgens available for target sites (72-78), in spite of normal plasma androgen levels. It is however not clear if these reduced SHBG plasma levels are due to a reduced synthesis of the protein, to the increased entry into target cells (79), or to increased binding to target cell membranes. A primary defect in the androgen binding capacity of SHBG was hypothesized showing that in some cases of hirsutism the Ka of DHT for SHBG was higher than in controls, leading us to suppose a qualitative defect of this protein increasing the peripheral androgen effect at skin level (80). Reviewing these data in the light of the recent discoveries of Rosner group (60) the increased Ka in these cases could amplify the transmembrane effects of the steroid-SHBG complex increasing the intracellular signals. Hyperinsulinemia may directly contribute to hyperandrogenism (81) by suppressing serum SHBG, thereby increasing the availability of free T to the skin. Moreover it is well known that insulin regulates fat metabolism but the influence of lipids on SHBG is still an argument of debate. Previous studies have suggested that plasma levels of free fatty acid (FFA) at a concentration within the normal physiological range can regulate free thyroid hormone levels (82, 83), the biologically available estradiol fractions in plasma (84) and also can inhibit T binding to albumin and SHBG increasing free T and modestly free DHT (85). Mendel's studies reject these observations and conclude that FFA are unlikely to affect interactions of steroids with their specific
Plasma markers Plasma markers of increased peripheral androgen action are rapresented by 3a androstanediol (3Ad) and 3a androstanediol glucuronide (3AdG). In idiopathic or peripheral hirsutism these parameters are the only ones plasma androgens increased (52, 53, 94-98) reflecting the increased skin formation of DHT, which on the contrary is an inadequate marker (54). In 1982 our group first suggested that unconjugated 3Ad could be a marker of idiopathic hirsutism which we defined "peripheral" to stress the ethiology of this form of hirsutism in which glandular androgens are within the normal range in several evaluations during the menstrual cycle (96). A previous study of Meikle (99) reported similar findings. In the same year Horton (94) showed that 3AdG, increased in 24/25 patients with idiopathic hirsutism, could be considered as a marker of skin androgen action, confirming the pioneer study of MauvaisJarvis showing that testosterone was a better precursor of urinary 3Ad when given through the skin than when given directly into the circulation (100). Previous studies have shown that 3Ad and 3AdG
arise from extrasplanchnic sites (101,102) and direct secretion has been excluded (103). Horton (94) proposed that 3AdG is formed in the skin analogously to other androgen target tissues (such as the prostate, in which a synthesis of 3AdG has been shown by the study of Chung and Coffee, 104). These data are largely confirmed by numerous studies published during the last five, six years (98,105-108). Three papers (109-111) however reported data showing that 3AdG cannot represent a good marker of idiopathic hirsutism and in two of those (109,110) androsterone glucuronide is proposed to be a better index of increased skin Sa reductase activity. In our opinion the first problem affecting these results is a still unclear definition of idiopathic hirsutism and secondly the selection of patients. Moreover the duration of hyperandrogenic status at the time when hirsute patient undergo the study is also an important variable which is not always taken into consideration. Idiopathic hirsutism may in fact convert to adrenal and/or ovarian hirsutism if untreated (112). An interesting form of hair growth is represented by prepubertal isolated hypertrichosis. In a group of affected girls in pre-adrenarcal age we demonstrated an isolated increase of DHT whereas 3Ad, 3AdG, SHBG , FT and T/SHBG ratio were within the normal range (113). These data suggested that increased Sa reductase at the hair follicular level could be a primary cause leading to this form of hair growth. In these cases plasma DHT, which is probably not locally further metabolized for an immaturity of the enzymatic machinery responsible for further DHT metabolism , reflect the increased 5a reductase activity. The role (direct or mediated by environmental growth factors) of increased local DHT have to be elucidated. Other possible metabolic pathways of DHT at skin level has been also postulated. A possibility that skin could produce C-19 sulpho-conjugates has been investigated by Matteri (114) finding that the formation of 3AdS from DHT is greater than that of 3AdG. Montalto (115) in a recent report concludes that 3AdS may be a marker of peripheral androgen metabolism evaluating this compound in patients affected by idiopathic hirsutism. Although several factors may be involved in the pathogenesis of acne, androgens are known to play an important and possibly a central role in the process. The Lookingbill study (116) showed in a group of women with mild to moderate acne, without hirsutism and with normally secreted androgens, significantly increased plasma levels of DHT and 3 AdG. This author concludes suggesting that
this form of acne is a result of a primary process involving increased 5a reductase activity at sebaceous glands level, confirming the role of 3AdG as a marker of peripheral androgen utilization. We were unable to confirm these results (117 , 118) in a larger number of women with mild or moderate acne and with secreted androgens within the normal range. In none of these cases we found increased levels of 3Ad or 3AdG or DHT, in spite of reduced levels of SHBG and increased values of FT comparable to values found in a parallel serie of idiopathic hirsute women, showing on the contrary increased plasma values of both 3Ad and 3AdG. On the basis of these results we conclude assuming that DHT in acneic patients could undergo to different metabolic pathway at a skin level with respect to hirsute patients , explaining the different clinical manifestations in front of similar androgen substrate . Glandular factors
a. Adrenal factors. Hirsutism represents one of the most frequent symptoms in Cushing's syndrome, which is however easily diagnosed for the concomitance of other pathognomonic symptoms like moon facies, weight gain with abnormal adipose distribution and striae. A mild congenital enzymatic defects in steroidogenesis could be a primary cause of hirsutism of adrenal origin. These forms differ from the classic congenital adrenal hyperplasia because the symptoms generally presented at puberty (119-126) or could be the cause of precocious pubarche (127) . This last symptom is frequently reported in the anamnesis of hirsute adult patients diagnosed as suffering of this form of adrenal hyperplasia due to 21-hydroxylase deficiency(personal observation). The pathogenesis of increased adrenal androgen secretion in these cases is secondary to the reduced production of cortisol leading to an increased secretion of ACTH or other pituitary factors which overstimulate androgen secretion from zona reticularis. These forms in which hirsutism appears at puberty are defined late onset or non classic congenital adrenal hyperplasia. The most frequent form is that due to 21-hydroxylase defect which presents high plasma levels of 17hydroxyprogesterone increasing following ACTH (0.2S0 mg e.v .). Non classic 21-hydroxylase deficiency, similarly to the classic form, is transmitted by an autosomal recessive gene linked to HLA system (128) . Association with HLA B14; DR1 provides a genetic marker useful in distinguishing the non classic variant from the classic disease, associated with HLA 160
Bw47; DR7 . A formal study of the association between HLA-B14; DR1 and non classic 21-hydroxylase deficiency demonstrated over 70% linkage disequilibrium between these two alleles in several ethnic populations, including Italians (129, 130). Another form which is very rarely diagnosed as a cause of hirsutism is the non classic form of congenital adrenal hyperplasia due to 11-hydroxylase defect, probably because the determination of 11 deoxycortisol (the plasma marker of this form) is not usually measured in all cases of hirsute women with high levels of 17 hydroxyprogesterone. 17 hydroxyprogesterone is in fact increased in this form, in some cases overlapping the values observed in the cases due to non-classic form of 21 hydroxylase defect (131-133) . A nonclassic form of 3B -01hydroxysteroid dehydrogenase defect as a cause of hirsutism was originally believed to be a very rare form. However, in recent years an increasing number of cases have been reported (124, 125, 134, 135), although the criteria to be followed for a correct diagnosis are still debated. The evaluation of 17 hydroxypregnenolone (17PRGN) and dehydroepiandrosterone and the ratios between these compounds which preceed the enzymatic defect and the 4-ene products, 17 hydroxyprogesterone (170HP) and androstenedione, both in baseline conditions and following ACTH could represent diagnostic markers. However the observation of the data reported in literature showed a large overlap between normal and hirsute patients considered affected by these defects. No linkage with HLA or other genetic marker have been documented. Only genetic studies sequenciing the genes coding for this enzyme should provide diagnostic instruments to ascertain the enzymatic defect. In the meantime diagnosis can be made only on the basis of the high values of at least the two ratios (17PREGN/170HP and DHA/A) which must increase following ACTH to confirm the diagnostic suspicion. However, it is important to consider that it has been demonstrated in animals that the prolonged exposition to high levels of androgens can reduce this enzymatic activity (136), leading to a plasma pattern which closely resemble that observed in 3B-ol- dehydrogenase defect. Therefore one should be very careful in considering hirsute patients, showing a long history of hyperandrogenism, affected by this defect (Fig. 4) . Adrenal androgen secreting tumors are very rare as a cause of simple hirsutism. Virilization usually occurs as a consequence of the very high levels of plasma testosterone (more than 150-200 ng/dl), which playa role in these cases. High levels of DHAS are frequently associated. If this hormonal pattern
NC CAH 210H
CORTI~L • AC Ht
ANDRO. ENS i Plasma Markers
0;~H~ ~PG~ DHA) \
Fig A - Non classical Congenital Adrenal Hyperplasia (NC CAH) - Pathogenetic scheme and plasma markers 21OH021 hydroxylase defect 38 01:38 hydroxysteroid dehydrogenase defect 118'11 hydroxylase defect 170HP: 17hydroxyprogesterone 17PGN'17hydroxyprogesterone OHA:dehydroepiandrosterone S:deoxycortisol
is found, radiological investigation is mandatory (137). b. Gonadal factors. A short report of 1983 (138) entitled "Polycystic ovary syndrome - what is it?" outlines the fact that the term polycystic ovary syndrome well fits the description of a clinical but not specific pathophysiologic syndrome, since several and different abnormalities could induces a similar clinical picture. Although we know much more about the physiopathology of normal and abnormal folliculogenesis, seven years later the question remains valid and the problem is still debated. The most widely accepted theory of the pathogenesis of the polycystic ovary syndrome is that this disease results from a complex cycle of events in which overproduction of androstenedione and its peripheral conversion to estrone are central (139, 140). In a very recent and excellent review Barnes and Rosenfield (141) proposed a new and simple theory. The syndrome arises from heterogeneous disorders that directly increase one of the three variables: the ratio of the serum concentrations of LH/FSH, the ratio of intraovarian concentrations of androgens/estrogens, or the process of follicular atresia. An increase in one of these variables, as a consequence of a primary defect in that area, may modify the other two in a cyclic manner. It is not clear how the pituitary secretion of go-
nadotropins or their hypothalamic control could be abnormal as a primary event leading to polycystic ovary syndrome (PCOS), it is much more plausible that the abnormal secretion occurs secondarily to an altered steroid feedback (139,140). Increased intraovarian androgens/estrogens ratio could derive from : 1. direct intraovarian increased production of androgens secondary to a genetic defect of intraovarian steroidogenesis or increased stimulation of androgens synthesis by insulin or other growth factors; 2.increased extraovarian androgen production. Several enzymatic defects have been found in PCOS patients : partial block in 3B hydroxysteroid dehydrogenase (142, 143), or in 17ketosteroid-reductase (144, 145), a presence of an abnormal 11 -hydroxylase (146), which may be associated with a presence of adrenal rest cells in the stromal tissue of the ovary (147), and very recently an intrinsic defect in P450c17 in ovarian thecal cells of patients with PCOS has been also postulated (148). Insulin and insulin-like growth factors has been shown to augment the response of theca cells to LH stimulation (149, 150) in vitro and a direct correlation between plasma androgen values and basal or glucose-stimulated insulin have been reported (151-154) in vivo, leading to the supposi-
tion that insulin may playa role in potentiating androgen production by the ovary. Hyperinsulinemia, particularly in obese patients, could therefore contribute to the abnormal androgen production in PCOS . Histopathological changes of the PCOS has been documented also as a consequence of an increased extraovarian androgen production (155) , as in adrenal congenital hyperplasia both classic (156) and non classic (157) or in untreated peripheral hirsutism (112) or in Cushing's syndrome . However, it is still not clear if androgens per se could be the cause of the altered gonadotropic pattern or if that it is the consequence of their peripheral estrone transformation . Otherwise it is well known that increased androgens could cause follicular atresia, blocking follicular maturation and antagonizing estrogen effects , resulting in anovulation (157). If androgens antagonize follicular maturation directly or by the means of increased intraovarian growth factors like EGF or TGF alpha , as suggested by Franks in a very interesting review (158), it warrants further study . Atretic follicles of PCOS patients has been documented to produce amounts of inihibin similar to viable follicles (159), further reducing FSH secretion in this syndrome. It
SKIN 5 alpha reductase
LIVER? 5 alpha reductase
5 alpha dihydrocortisol
ADRENALS PR Adrenal Androgens
Fig.5 - Polycystic ovary syndrome Pathogenetic scheme and plasma markers. OHT:dihydrotestosterone 3 Ad:3a androstanediol 3 AdG:3a androstanediol glucuronide
Increased androgens/estrogens ratio - Increased intraovarian androgens: - 17KS, P45017 defects - Increased production by insulin, IGF-1 - Increased extraovarian androgen production - Adrenals, skin Increased follicular atresia - Androgens effect (direct or indirect?) - Decreased follicular maturation by EGF or aTGF?
LH/FSH i Androstenedione Estrone i
has been very well documented that in animals the exogenous supplementation of androgens could induces polycystic ovarian changes at histological observation (160,161). Of particular interest is the very recent paper (162) in which the authors suggested that PCOS may be a disease of the liver and the skin. They based their conclusions on the fact that PCOS patients presented increased 5a reductase activity both in the skin and in the liver. This increase in the skin causes an augment of dihydrotestosterone formation leading to hirsutism. The increase in the liver augment cortisol clearance, being cortisol metabolized to 5a dihydrocortisol. The increased metabolic clearance of cortisol (supported by the findings of increased urinary excretion of cortisol metabolites) could cause the increase in corticotropin secretion to keep plasma cortisol concentrations normal but at expense of androgen excess. This theory strictly correlates with our previous paper (109) in which we showed progressive clinical and hormonal modifications in untreated hirsute patients during the natural history of the disease. In this paper we conclude affirming that hirsutism could be an evolving syndrome rather than a static condition, initially involving increased skin formation of DHT. At a later step adrenal cortex may be involved and finally alterations in ovarian function may develop possibly mediated by changes in hypothalamic-pituitary responsiveness. These two studies outline the central role of skin altered metabolism in all clinical manifestations presenting hirsutism and try to explain how these peripheral alterarations can involve glandular androgen production (Fig. 5). In an attempt to find an experimental support for this theory we have treated guinea pigs which are appropriate model in this kind of study, with high doses of DHT. Results showed an isolated growth of zona reticularis of the adrenal gland, documented by morphometric analysis, and increased circulating levels of 5ene steroids (136). Studies are in progress to evaluate the ovarian modifications and to understand how DHT can induce these morphological and biochemical modifications. Another cause of ovarian hyperandrogenism is represented by the androgen secreting tumors which, like adrenal tumors, are characterized by rapid evolution of hirsutism of a high degree and usually present virilization as a consequence of very high plasma values of T (more than 150-200 ng/dl). The dynamic test is not useful in these cases to establish the origin of hyperandrogenism (163) and diagnostic suspicion has to be confirmed by radiologic investigations.
Summarizing our opinion, we believe that hirsutism in the majority of cases has to be regarded as a skin disease. Glandular implication could be a consequence of the increased 5a reductase activity with augmented steroid clearance, which in turn could modify the hypothalamic-hypophyseal response. Other factors (such as obesity, hyperinsulinemia, growth factors) could interfere both at skin and at glandular levels complicating the pathogenetic mechanism. The difference observed in plasma hormonal pattern in hirsute patients and the response to therapy depends on the time elapsed between the beginning of symptoms and the patient's observation. Congenital enzymatic defects in steroidogenesis or dysregulated activity of citochromes and primitive androgen secreting tumors both of adrenals or ovaries could represent only the primitive glandular causes of hyperandrogenism. AKNOWLEDGMENTS I should like to thank all my collaborators who contribute to my work in the field, argument of this review: Maria Vittoria Adamo, Sonia Foli, Stefani a Caiola, Marella Maroder, Daniela Casilli, Adele Mangiantini and Patrizia Bianchi. I am most grateful to my colleague Rina Balducci for the helpful suggestions in the preparation of the manuscript.
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