Insulin Action and Its Disturbances in Disease - part 9 docx

DEFINITION OF POLYCYSTIC OVARY SYNDROME PCOS 487INSULIN OBESITY INSULIN RESISTANCE PITUITARY ADRENAL GLANS P450c17 ANDROGENS DHEAS PCOS OVARY P450c17 ANDROGENS TESTOSTERONE ANDROSTENEDIO

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36 Cleland, S J., Petrie, J R., Small, M., Elliott, H L and Connell, J M C (2000) Insulin action is associated with endothelial function in hypertension and type 2 diabetes.

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37 Abe, H., Yamada, N., Kuwaki, T., Shimada, M., Osuga, J., Shionoiri, F., Yahagi, N., Kadowaki, T., Tamemoto, H., Ishibashi, S., Yazaki, Y and Makuuchi, M (1998) Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular

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61 Peraldi, P., Xu, M and Spiegelman, B M (1997) Thiazolidinediones block tumor

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63 Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z W., Karin, M and Shoelson, S E (2001) Reversal of obesity- and diet-induced insulin resistance with

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66 Stears, A J and Byrne, C D (2001) Adipocyte metabolism and the metabolic syndrome.

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67 Saltiel, A R and Kahn, R C (2001) Insulin signalling and the regulation of glucose and

lipid metabolism Nature 414, 799 – 806.

68 Shulman, G I (2000) Cellular mechanisms of insulin resistance J Clin Invest 106,

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69 Bermudez, E A., Rifai, N., Buring, J., Manson, J E and Ridker, P M (2002) Interrelationships among circulating interleukin-6, C-reactive protein, and traditional

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70 Han, T S., Sattar, N., Williams, K., Gonzalez-Villalpando, C., Lean, M E J and Haffner, S M (2002) Prospective study of C-reactive protein in relation to the development of diabetes and metabolic syndrome in the Mexico City Diabetes Study.

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71 Festa, A., D’Agostino, R., Tracy, R P and Haffner, S M (2002) Elevated levels of acute-phase proteins and plasminogen activator inhibitor-1 predict the development of

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72 Bastard, J.-P., Maachi, J., Van Nhieu, J T., Jardel, C., Brucker, E., Grimaldi, A., Robert, J.-J., Capeau, J and Hainque, B (2002) Adipose tissue IL-6 content correlates

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73 Mantzoros, C S (1999) The role of leptin in human obesity and disease: a review of

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76 Nagaev, I and Smith, U (2001) Insulin resistance and type 2 diabetes are not related to

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The most frequent forms of hyperandrogenism are premature pubarche fined as the appearance of pubic hair before 8 years) in the pre-pubertal periodand PCOS in the post-pubertal period, which affects approximately 5–10 percent of women of reproductive age.2

(de-Insulin resistance and compensatory hyperinsulinaemia are prominent tures of many women with PCOS The aetiology of this condition is unknown,but recent evidence suggests that the principal underlying disorder is insulinresistance and that the resulting hyperinsulinaemia stimulates excess ovarianandrogens.3 Associated with insulin resistance, these women exhibit hyperlip-idaemia and have a high risk of type 2 diabetes and cardiovascular disease inlater life.4, 5The new concept arising from the link with insulin resistance intro-duces a concept that not only has major implications for the health of affectedwomen but also offers a potential for new treatments

fea-Nowadays, the current treatment mainly with antiandrogens has been ated with insulin sensitizers such as metformin or thiazolidinediones The resultsobtained with these drugs seem to confirm their efficacy in reversing metabolicand ovarian abnormalities in these women and adolescent girls

associ-Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly

 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6

16.2 Definition of polycystic ovary syndrome (PCOS)

and diagnostic criteria

PCOS is probably the most common endocrine disorder in women Althoughnot universally accepted, the 1990 point Conference of the National Institute ofHealth/National Institute of Child Health and Human Development establishedthe diagnostic criteria on PCOS

PCOS is defined as a metabolic condition characterized by hyperandrogenism(hirsutism, acne, androgenic alopecia) and chronic anovulation (irregular menseswith menses every 6 weeks to 6 months or amenorrhea) with the exclusion of spe-cific disorders, such as non-classical adrenal hyperplasia due to 21-hydroxylasedeficiency, hyperprolactinaemia, androgen-secreting tumours and thyroid dis-eases Thus, the most widely accepted definition of PCOS is the association ofclinical and/or biochemical evidence of androgen excess with chronic anovulation(having excluded specific underlying disorders of the pituitary or adrenals).6

This syndrome as a form of functional ovarian hyperandrogenism is a lent disorder affecting approximately 5–10 per cent of reproductive women.2

preva-The prevalence of polycystic ovaries increases throughout puberty, reachingabout 26 per cent by the age of 15 The prevalence of PCOS among teenagegirls is not known but is clearly common.7

Ethnic differences in the prevalence of PCOS have not been explored butnot significant differences between white and black women in the USA havebeen observed.2 Similar prevalence (6.8 and 6.5 per cent) was reported in twoEuropean countries.8, 9

Insulin resistance, a common feature of PCOS, can be characterized as paired action of insulin in the uptake and metabolism of glucose Impairedinsulin action leads to elevated insulin levels, which causes a decrease in thesynthesis of two important binding proteins: insulin-like growth factor bindingprotein (IGFBP1) and sex hormone binding globulin (SHBG) IGFBP1 bindsIGF I and IGF II and SHBG binds to sex steroids, especially androgens Obesity,which is seen in 50–65 per cent of PCOS patients, may increase the insulinresistance and hyperinsulinaemia.3

im-Acanthosis nigricans, a dark and hyperpigmented hyperplasia of the skin ically found at the nape of the neck and axila, is a marker of insulin resistance.Acanthosis nigricans is usually found in about 30 per cent of hyperandrogenicwomen The triad of hyperandrogenism, insulin resistance and acanthosis nigri-cans (HAIR-AN) syndrome appears in a subgroup of patients with PCOS.10

typ-Chronically elevated luteinizing hormone (LH) and insulin resistance are two

of the most common endocrine aberrations seen in PCOS The genetic cause of

high LH is not known In vitro and in vivo evidence offers support that high LH

and hyperinsulinemia work synergistically, causing ovarian growth, androgenproduction and ovarian cyst formation.1 Figure 16.1 shows the multiple factorsthat can contribute to the development of PCOS

DEFINITION OF POLYCYSTIC OVARY SYNDROME (PCOS) 487

INSULIN

OBESITY

INSULIN RESISTANCE PITUITARY

ADRENAL GLANS

P450c17

ANDROGENS DHEAS

PCOS OVARY

P450c17

ANDROGENS TESTOSTERONE ANDROSTENEDIONE

AND FOLLICLE ARRESTANOVULATION

OTHER FACTORS

Genes

Puberty

Premature pubarche

The diagnosis of polycystic ovary syndrome is usually made on the basis of

a combination of clinical and biochemical criteria (Table 16.1)

The degree of hirsutism can be assessed by the Ferriman–Gallwey score, asimple, semiquantitative method for recording the distribution and severity ofexcess body hair.11

The classic anatomical pattern of polycystic ovaries can be identified byultrasound assessment as increased number of subcapsular follicular cysts andincreased intervening stroma.12These ultrasound features are consistent with, butnot essential for, the diagnosis of the syndrome.13 Serum levels of testosteroneand androstenedione are usually increased DHEAS dehydroepiandrosterone sul-fate levels are increased by up to 50 per cent in women with PCOS Elevatedfree testosterone activity, defined by the free androgen index, represents themost sensitive biochemical marker supporting the diagnosis Prolactin is usually

Table 16.1 Clinical and biochemical evaluation of PCOS

Menstrual disturbances Ferriman – Gallwey score Clinical Pelvic ultrasonography

Obesity (BMI) Testosterone, androstenedione, DHEAS SHBG

No single test is diagnostic of the syndrome, but choice should be guided

by clinical presentation Serum LH levels are typically elevated in PCOS but

up to 50 per cent of the young women with other clinical and biochemicalfeatures of the syndrome may have normal serum LH levels Measurement of

LH is therefore of limited diagnostic value; it is quite specific that raised LHand normal FSH essentially occur only in PCOS, but this is not very sensitive.1

To assess insulin resistance with compensatory hyperinsulinism, fasting bloodglucose and insulin could be useful and simple to detect a primary abnormal-ity With a standard oral glucose tolerance test, a hyperinsulinaemic response,impaired glucose tolerance or type 2 diabetes could be documented

The abnormal response of 17 α-hydroxyprogesterone after an agonist logue of gonadotrophin-releasing hormone (GnRH) challenge has been described

ana-in women and adolescents.15, 16 Short-term leuprolide acetate (500 µg sc) is areliable tool for identification of the ovary source of hyperandrogenaemia Theresponse was considered supranormal if the peak plasma 17α-hydroxyprogester-one 24 h postestimulation was greater than 4.75 nmol/l (160 ng/dl).16

Hyperandrogenism in PCOS may therefore represent an intrinsic abnormality

of ovarian theca-interstitial cell function This conclusion is supported by clinicalstudies suggesting that the ovary is the primary abnormality site

The response observed in women with PCOS in the above mentioned test(GnRH agonist) could not be explained on the basis of LH hyper-responsiveness.Women with PCOS given an hCG challenge test produce more androstenedioneand 17α-hydroxyprogesterone than normal subjects and this difference remainsevident after suppression of endogenous LH secretion by GnRH.17, 18

As many hyperandrogenic anovulatory women have significantly increasedovarian steroidogenic responses to stimulation with GnRH analogues, Rosenfieldand colleagues have coined the term ‘functional ovarian hyperandrogenism,’ as

an alternative to PCOS.19

HYPERANDROGENISM AND HYPERINSULINISM 489

The earliest description of ‘diabete des femmes a barbe’ pointed out the tionship between androgen excess in women and disturbances in carbohydratemetabolism.20The coexistence of severe insulin resistance and acanthosis nigri-cans in three lean adolescent women confirmed the association between hyper-androgenism and hyperinsulinism.21

rela-Insulin resistance associated with PCOS was also reported some years later byChang and colleagues in 1983.22This resistance, which is independent of obesity,causes hyperinsulinaemia23and more than 50 per cent of the obese women withPCOS are insulin resistant compared with age and weight-matched controls.24

Hyperinsulinaemia is shown to be a characteristic finding in women withovarian androgen excess, even in the absence of diabetes Nowadays, it hasbecome evident that insulin resistance is a cardinal feature of PCOS that couldserve as the pathogenic link between hyperandrogenism and hyperinsulinism.Because insulin resistance is related to many manifestations of PCOS, theretends to be substantial overlap between the PCOS phenotype and the so-called

‘metabolic syndrome’ or ‘syndrome X’: obesity, glucose intolerance, sion, macrovascular disease and dyslipidaemia, which are seen in both syn-dromes Figure 16.2 shows the metabolic and endocrine disorders associatedwith PCOS and insulin resistance

hyperten-It is generally accepted that women with PCOS are predisposed to type 2diabetes and that the development of diabetes cannot be attributed solely to theobesity that typically accompanies PCOS The prevalence of impaired glucosetolerance in PCOS is between 30 and 40 per cent and that of type 2 diabetes

is between 5 and 10 per cent.4, 5 These prevalences approximate those in Pimaindians, who have one of the highest rates of diabetes in the world In addition,suggesting some genetic risk factor in this process, most women and adolescentswith PCOS have a family history of type 2 diabetes.25, 26

Elevated serum androgens may at times cause mild insulin resistance but it isunlikely that the insulin resistance of PCOS occurs as a result of hyperandrogen-ism.27 Insulin resistance persists in women with PCOS in whom both ovarieshave been removed surgically or in women whose ovarian androgen produc-tion has been suppressed with the use of long-acting gonadotrophin-releasinghormone (GnRH) agonist.1

Pre-pubertal women with acanthosis nigricans are hyperinsulinaemic, yet vated serum androgen levels do not appear until several years following thediagnosis of insulin resistance In the same way, some women with point muta-tions in the insulin receptor gene causing hyperinsulinaemic insulin resistancehave been shown to have PCOS Collectively, the genetic syndromes of severeinsulin resistance secondary to mutations in the insulin receptor gene (leprechau-nism, Rabson–Mendenhall syndrome and type A insulin resistance syndrome)have a common phenotype characterized by hyperandrogenism, insulin resis-tance with hyperinsulinism and acanthosis nigricans These observations support

ele-PCOS Insulin resistance

Androgen production

Metabolic disorders

Endocrine disorders

Obesity

Figure 16.2 Endocrine and metabolism disorders in PCOS

the idea that the hyperinsulinaemia of PCOS is a causal factor in the nying hyperandrogenism.3

accompa-It has been suggested that insulin, as IGFI, is capable of enhancing a variety ofsteroidogenic pathways, not only in ovarian thecal cells, but in ovarian granulosacells, adrenocortical cells and the periphery Furthermore, insulin seems to becapable of exerting such effects directly, at elevations too modest to invoke suchaction via interaction with the IGFI receptor.28

Hyperinsulinaemia appears to be a major factor in the ovarian dysfunction ofPCOS Any treatment that lowers insulin levels produces a decrease in androgenlevels and improves ovarian function

The increase in insulin levels common in PCOS may precipitate androgenaemia in genetically vulnerable individuals by acting through latentabnormalities in steroidogenesis regulation, although it probably has only aminimal effect on ovarian function in many individuals

hyper-Paradoxically, hyperinsulinaemia is capable of exerting systemic effects inpatients moderately resistant to the effects of insulin on glucose metabolism.Thus, it is capable of lowering IGFBP1 and SHBG concentrations and stimu-lating ovarian steroidogenesis

ASSESSMENT OF INSULIN RESISTANCE IN PCOS 491

One plausible hypothesis that tries to explain the relationships between insulinaemia and hyperandrogenaemia is the unified serine activity in bothinsulin receptors and cytochrome P450c17 Hormonally regulated serine phos-phorylation of adrenal P450c17 by a c-AMP-dependent kinase accounts for alarge increase in 17–20 lyase activity and has been proposed as the mecha-nism for normal adrenarche.29 Phosphorylation studies of the insulin receptors

hyper-in fibroblasts from PCOS patients have shown that around half of the PCOSwomen have an increase in serine phosphorylation, which produces an inhibi-tion of tyrosine phosphorylation and a reduction of insulin signal transduction.30

This means that abnormal serine phosphorylation, possibly associated with asingle kinase, may be responsible for excessive serine phosphorylation of theinsulin receptors and P450c17, leading to insulin resistance and adrenal/ovarianhyperandrogenism Even though the responsible kinase has not been identifiedand the explained theory has not been confirmed, recent results suggest that aserine kinase-mediated pathway may be involved in the insulin resistance ofPCOS patients.31

At the moment, there is no unified theory to explain a heterogeneous ease such as PCOS, but the key role of insulin in this process is not ques-tioned (Figure 16.1) The onset may occur in late childhood since many of themetabolic and endocrine features of the disorder mimic puberty Associatedwith this are increases in the pulse, an amplitude of luteinizing hormone (LH),increasing androgen concentrations, hyperinsulinism and irregular menses Mul-tiple, small ovarian cysts are seen on ultrasound examination and are a commonand normal feature of puberty It is therefore possible that women geneticallypredisposed to polycystic ovarian syndrome fail to resume normal insulin sensi-tivity and continue to express metabolic and endocrine features usually confined

dis-to puberty.32, 33

16.4 Assessment of insulin resistance in PCOS

The euglycaemic–hyperinsulinaemic clamp technique34is the gold standard forassessing insulin sensitivity and it is often combined with the hyperglycaemicclamp to determine the adequacy of compensatoryβ-cell hypersecretion.35

Insulin resistance andβ-cell responsiveness can also be assessed by the quently sampled intravenous glucose tolerance test.36

fre-However, they cannot be used on a routine clinical basis or epidemiologicalstudies because they are too laborious, time consuming and invasive, especially

in children and adolescents Surrogates based on fasting glucose and insulin and

on insulin and glucose responses to oral glucose have often been used

Although assessment of either fasting or peak insulinaemia after OGTT couldprovide sufficient data to classify individuals into normal, mild to moderate andsevere insulin resistance, the results of this test must be interpreted in the context

of plasma glucose levels, because the presence of any degree of hyperglycaemia

suggests the existence of defects in insulin secretion, which invalidates thedegree of insulinaemia as an index of insulin resistance Fasting insulin lev-els above 50–70µU/ml or insulin peak in post-oral-glucose challenge above

350 µU/ml suggest severe insulin resistance, in contrast to the fasting insulinlevels below 20µU/ml or OGTT peak insulin below 150 µU/ml observed innormal individuals.37

Various indices have been derived from the basis data provided by the oralglucose tolerance test (OGTT) which allow quantitative estimation of β-cellfunction, such as mean serum insulin index (MSI).38, 39

Measures of insulin sensitivity based on fasting glucose and insulin includethe homeostasis model assessment (HOMA),40 fasting insulin resistance index(FIRI),41fasting glucose insulin ratio,24and quantitative insulin sensitivity checkindex (QUICKI)42 and others

Determining the fasting glucose insulin ratio could be a good screening test

in that it is simple, quick and relatively inexpensive to obtain a single bloodsample, and it has been validated against ‘gold standard’ methodology.43, 44However, the glucose insulin ratio is most useful in a purely insulin-resistantpopulation, before overt β-cell dysfunction develops

A fasting glucose–insulin ratio of less than seven in girls with prematurepubarche or obesity may be helpful in the early identification of those at riskfor complications of insulin resistance43and this finding was recently validated44

by a stepwise regression analysis showing that the fasting glucose–insulin ratiowas significantly predictive of insulin sensitivity A ratio of less than seven is

a cut-off for diagnosis of insulin resistance in adolescents with PCOS, and inadult women with PCOS the ratio is less than 4.5.24 As mentioned, measures

of insulin sensitivity can also be obtained from the OGTT

Fasting insulin sensitivity and post-oral-glucose compensatory naemia are closely related, although they do reflect distinct aspects of glucoseregulation Fasting insulin levels reflect hepatic insulin sensitivity and the abil-ity of insulin to suppress hepatic glucose production.45Post-oral-glucose insulinexcursions, on the other hand, in part reflect the need to suppress hepatic glucoseproduction and also the requirement to increase peripheral glucose disposal.45

hyperinsuli-The high prevalence of impaired glucose tolerance and type 2 diabetes tus found in adult women with PCOS was also found in adolescents with PCOS

melli-by means of 2 h glucose levels after 75 g glucose challenge.46 To predict theseabnormalities the OGTT would be the choice and it was finally recommendedthat adolescents with PCOS should undergo periodic screening for abnormalglucose tolerance using 2 h post-challenge plasma glucose levels.47

Several reports have stressed that PCOS is a familial disorder; however, thegenetic basis of the syndrome remains controversial.48

GENE STUDIES ON PCOS 493

It is difficult to determine the mode of inheritance of this heterogeneoussyndrome and there is an absence of an equivalent male phenotype Some studieshave revealed an autosomal dominant mode of inheritance considering prematurebalding in men as the primary male phenotype.49, 50

On the other hand, there are studies of families with high prevalence of PCOS

in which the Mendelian autosomal dominant mode of inheritance cannot explainthe mode of inheritance of the syndrome,51 while in another study an X-linkedmodel was postulated.52 As a result, the mode of inheritance remains unclearand more than one gene defect seems to participate in the pathogenesis of thesyndrome Thus, PCOS appears to be an oligogenic disorder and several genesmay be involved in its aetiology

The presence of insulin resistance and compensatory hyperinsulinaemia led

to the assumption that genes involved in the secretion and action of insulin mayplay a role in the pathogenesis of PCOS

Molecular studies of the coding region of the insulin receptor gene in womenwith PCOS have shown a large number of silent polymorphisms, mainly inintronic regions The majority of these polymorphisms have also been iden-tified in normal subjects and are considered to be common polymorphisms,which do not lead to remarkable molecular disturbance in the insulin receptorgene.53

There is, however, evidence of a stable abnormality in insulin receptor phorylation in cells from women with PCOS Increased insulin-dependent serinephosphorylation of the insulin receptor β-subunit in skin fibroblast and skele-tal muscle from 50 per cent of the women with PCOS was found comparedwith controls.30 The serine-phosphorylated insulin receptor had reduced ability

phos-to phosphorylate tyrosine, suggesting that it may impair signal transduction

A single-nucleotide polymorphism in the exon 17 C/T of the insulin receptorwas most frequently found in lean patients with PCOS compared with leancontrols, but the role of this susceptibility needs to be determined.54

The minisatellite of the insulin gene INS VNTR (insulin gene variable numbertandem repeats) has been investigated since this region is directly implicated inthe regulation of insulin secretion

The INS VNTR is a functional polymorphism, so it regulates the transcription

of the insulin gene and probably the expression of the IGF-II gene, which isadjacent to the insulin gene.55An association between PCOS and allelic variation

at the INS VNTR locus has been reported It was shown that class III allelesand especially III/III genotypes are associated with PCOS and are most stronglyassociated with anovulatory PCOS The group of women with one or two classIII alleles had significantly higher fasting insulin levels and higher mean bodymass index than women with the I/genotype.56 This finding was confirmed

in another study.57 Conversely, in another European population of girls whopresented with precocious pubarche, hyperinsulinaemia and dyslipidaemia wererelated to both birth weight and INS VNTR class I alleles.58

Other candidate genes in the pathogenesis of PCOS are the encoding genes

of steroidogenic enzymes, such as CYP17, CYP11α and CYP19

Recent studies have shown that PCOS may be the result of overfunction of theenzyme that catalyses androgen production (cytochrome P450c17α) CytochromeP450c17α is an enzyme with two functions, since it has both 17 α-hydroxylaseand 17,20-lyase activities In the thecal cells P450c17α converts progesterone

to 17 α-hydroxyprogesterone through its 17 α-hydroxylase activity, and then itconverts 17 α-hydroxyprogesterone to androstendione through its 17,20-lyaseactivity.15

Clinical studies have shown an abnormality in the regulation of 17lase/17,20-lyase (the rate-limiting step in androgen biosynthesis in the ovariesand the adrenals) in women presenting with PCOS, as evidenced by increased

α-hydroxy-17 α-hydroxylase and to a lesser extent 17,20-lyase activity, since in thesewomen there is an exaggerated serum 17α-hydroxyprogesterone to stimulation

by gonadotrophin-releasing hormone agonists, as already mentioned.19

The other gene involved in the steroidogenic pathway is CYP11α, whichencodes P450scc, the enzyme for cholesterol side chain cleavage that catalysesthe conversion of cholesterol to pregnenolone, which is the initial and rate-limiting step at the start of the steroid hormone biosynthetic pathway It hasbeen hypothesized that up-regulation of this enzyme could lead to increasedandrogen production.59

After some contradictory results, no association was found between any ofthe alleles of the CYP11α and the presence of PCOS.60, 61

The enzyme aromatase encoded by CYP19 catalyses the conversion of gens to oestrogens It has been found that granulosa cells from anovulatorypolycystic ovaries are hyper-responsive to follicle-stimulating hormone (FSH)

andro-in vitro, displayandro-ing significantly greater oestradiol production than granulosa

cells from normal ovaries.62 So far, there is no evidence of any association ofalleles of this gene with PCOS.63

The androgen receptor, through which all androgens act, has also been tigated, especially the polymorphic CAG repeat within exon 1, which encodes apolyglutamine chain in the N-terminal transactivation domain.64 The length ofthe polymorphic CAG repeat sequence is inversely correlated to the androgenreceptor transcriptional activity

inves-An association between increased hirsutism and decreased CAG repeat lengthhas been demonstrated.65, 66 However, further studies need to be conducted to

analyse the role of androgen receptors in the pathogenesis of PCOS

Another gene studied to analyse the possible genetic origin of PCOS is LHβ-subunit gene; since about 50 per cent of women with PCOS have hyper-secretion of LH associated with anovulation, an adverse role of LH gene may

be suspected

One polymorphic variant seems to protect obese women from developingsymptomatic PCOS,67 but another LH variant has been identified as a result of

PREMATURE PUBARCHE, HYPERINSULINISM AND PCOS 495

a single missense mutation in exon 3 of the LH β-subunit gene This variantseems to play a role in female infertility but further studies are required todetermine the pathological significance of this variant.68

Recent investigations have shown an association between PCOS and

follistat-in,60 but the contribution of follistatin gene in the development of PCOS hasnot been confirmed.69, 70

Premature pubarche is defined as the early appearance of pubic hair, before 8years in girls and 9 years in boys, independently of the appearance of axillaryhair and apocrine secretion, and of pubertal development The incidence ofpremature pubarche is almost tenfold higher in girls than in boys In most cases,premature pubarche is due to an exaggerated variant of normal maturation ofadrenal gland function being a most frequent form of hyperandrogenism in thepre-pubertal period.71

Enzymatic defects of steroidogenesis are pathological causes of prematurepubarche, with a reported frequency around seven per cent in these girls.72

Genetic defects in the CYP21 gene, which encodes the 21 hydroxylase zyme, have been investigated, and the incidences of molecular defects were com-parable in the premature pubarche and control groups There is no relationshipbetween the presence of carrier status and endocrine–metabolic abnormalities.73

en-Prospective studies of larger cohorts of premature pubarche girls are needed toascertain the long-term clinical relevance of CYP21 heterozygosity

In the absence of an adrenal enzymatic defect, premature pubarche has beenassociated with an acceleration of statural growth and bone maturation, with-out affecting the timing of the onset or the progression of puberty or thefinal height.74

Re-evaluation of adrenal function in young women with a history of ture pubarche revealed an increased incidence of so called ‘idiopathic functionaladrenal hyperandrogenism’ A pattern of adrenal secretion that affects 50 percent of these girls gives rise to a suprahormonal response to ACTH test Idio-pathic functional adrenal hyperandrogenism has been attributed to a dysregula-tion of adrenal cytochrome P450c17, prominently in the
5 pathway.75

prema-Post-pubertal follow-up of girls with premature pubarche has documentedmore than tenfold prevalence of ‘functional ovarian hyperandrogenism’ (45versus 3 per cent in the normal adolescent population), a form of PCOS atadolescence, which is usually associated with hyperinsulinaemia and dyslipidae-mia.46, 76 This sequence seems to occur more frequently in girls with elevatedDHEAS and or androstenedione at diagnosis of premature pubarche.77

Assessment of ovulatory function in girls with a history of precocious arche revealed that the fractions of ovulating girls and ovulatory cycles inlate post-menarche were strikingly higher (P < 0.001) in the non-premature

pub-pubarche than in the premature pub-pubarche subgroup (91 versus 20 and 47 versus

12 per cent), with no differences in early post-menarche.78 It could be assumedthat the development of ovarian hyperandrogenism after premature pubarche ispreceded by an apparently normal phase, with regular cycles lasting for about3–5 years after menarche

In general, puberty is associated with increasing fasting and stimulated insulin concentrations and a decrease in insulin sensitivity.79, 33 Theinsulin resistance during puberty is restricted to peripheral glucose metabolismand is associated with concomitant increases in growth hormone and insulin-like growth factor (IGFI) secretion and a decrease in IGFBP1 and SHBGconcentrations.80

glucose-The hyperinsulinaemia and increased IGFI activity during puberty havebeen proposed as inducing factors in the development of PCOS in susceptiblesubjects.32, 81

In girls with premature pubarche, hyperinsulinism is already detectable beforepuberty and throughout all states of pubertal development It is often accom-panied by an increased early insulin response to glucose, by an elevated freeandrogen index and by decreased IGFBP1 and SHBG concentrations.46 In addi-tion to hyperinsulinaemia, girls with premature pubarche display supranormaltriglyceride levels, very low density lipoprotein cholesterol, and very low densitylipoprotein triglyceride concentrations.76

Both hyperinsulinaemia and altered lipid profile support the concept that thecluster of highly atherogenic abnormalities may already start by childhood, inagreement with other studies pointing towards an early development of the patho-physiological events leading to type 2 diabetes and cardiovascular disease.82The frequent association of premature pubarche with functional ovarian hyper-androgenism and hyperinsulinism could have in common early origin rather thanbeing the result of a direct inter-relationship later in life Reduced foetal growthwas first related to type 2 diabetes in older adults83 and also was found to beassociated with insulin resistance in pre-pubertal and post-pubertal children bornsmall for gestational age.84, 85

Girls with premature pubarche have lower birth-weight standard deviation(SD) scores than control girls.86 Those girls with premature pubarche who sub-sequently develop functional ovarian hyperandrogenism have even lower birthweights Finally, the lowest birth weights were found in girls with – in addi-tion – pronounced hyperinsulinism86(Figure 16.3)

The precise mechanism governing the aforementioned relationship is currentlyunknown, but the results seem to suggest that premature pubarche and hyperinsuli-naemia may precede the development of ovarian hyperandrogenism, and possiblyPCOS, and that this sequence may have a common early origin (low birth weightserving as a marker) These data support the early life hypothesis that disease inpost-natal life may have its origin in the foetal environment, and that this processcan be attributed to changes in the programming of foetal endocrine axes.83

TREATMENT APPROACH WITH ANTIANDROGENS 497

+

n = 25 – –

n = 11 +

+ + +

n = 12 – +

post-The follow-up related findings in girls with premature pubarche suggest thatthis process should no longer be considered a normal variant of development butrather a clinical marker of endocrine–metabolic disorders associated with reducedfoetal growth

16.7 Treatment approach with antiandrogens

Current treatments until now have been addressed to reduce the main presentingfeatures such as irregular menses, hirsutism or infertility Oral contraceptives arecommonly used to regulate menses and decrease ovarian androgen production.Increasing levels of sex-hormone-binding globulin while decreasing ovarianandrogen production reduces the circulating free testosterone and subsequentlyandrogen activity; however, the combined pill exacerbates insulin resistance,mainly in obese patients, in whom this treatment may be unsuitable

Hirsutism may be addressed by the use of antiandrogens, cyproterone acetate

or spironolactone Their principal mode of action is the inhibition of the binding

of dihydrotestosterone to its receptors at the hair follicle Beneficial effects can beseen after some months of treatment, but excessive hair growth returns soon aftercessation of treatment Cyproterone acetate may exacerbate irregularity of themenstrual cycle, and both drugs are unsuitable for use in those trying to conceive.Another alternative approach to be used is finasteride, an inhibitor of the type 2isoenzyme of 5α-reductase, the enzyme responsible for conversion of testosterone

to the active metabolite dihydrotestosterone The other one is flutamide, the mostcommon antiandrogen used as a therapeutic regime in the treatment of hirsutism.Flutamide is a non-steroidal compound that seems to act only at the receptorsite and is therefore considered a pure antiandrogen Liver toxicity is a rare butpotentially severe side-effect of flutamide, which is dose dependent.

Several recent papers have been published evaluating treatment results of PCOSwith flutamide, spironolactone, cyproterone acetate, ketoconazole and finaster-ide.87 – 89 Those drugs employed currently constitute a satisfactory alternativetherapeutic regime in the treatment of hyperandrogenism However, a long treat-ment period is always required to improve hirsutism and prevent or delay itsrelapse.87The reduction of androgen levels by flutamide restores normal ovarianregulation of GnRH secretion in PCOS and may have a place in the therapeuticregime aimed at establishing cyclic ovulation in women with PCOS.89

According to our results,88flutamide treatment was accompanied by a markeddecrease in hirsutism score, free androgen index, testosterone and androstene-dione levels and by an increase in sex-hormone-binding globulin concentrations.However, there were no substantial changes in the pattern of menstrual cycles,gonadotropin, oestradiol or dehydroepiandrosterone sulfate concentrations, andthere were no detectable effects on the 17-hydroxyprogesterone response to GnRHagonist Serum triglycerides, total cholesterol and low-density lipoprotein choles-terol levels decreased markedly during flutamide therapy, whereas high-densitylipoprotein cholesterol, fasting glycaemia–insulinaemia and the insulin response

to a glucose load remained unchanged

In conclusion, low dose flutamide treatment was found to be an effective andsafe approach to reduce hirsutism and circulating androgen, low-density lipopro-tein cholesterol and triglyceride levels in girls with functional ovarian hyper-androgenism after premature pubarche.88 However, flutamide failed to increasehigh-density lipoprotein cholesterol levels or decrease hyperinsulinaemia, thesebeing two major risk factors for subsequent cardiovascular disease

16.8 Treatment approach with insulin sensitizers

(metformin)

Taking into consideration the aforementioned, the administration of insulin tizer drugs such as metformin or thiazolidinediones could potentially reverse themetabolic process and restore the ovarian function

sensi-Several reports have been published in recent years addressing evaluation ofthe effect of insulin sensitizer agents in PCOS women, such as biguanides andthiazolidinediones (metformin and troglitazone)

Most of the metabolic abnormalities of PCOS can be reversed by metformin,with the additional benefit of enough normalization of the endocrine milieu toallow regular menstrual cycles, reversal of infertility and spontaneous pregnancy.Thus, one report,90despite the short treatment period (8 weeks), was able to show

TREATMENT APPROACH WITH INSULIN SENSITIZERS (METFORMIN) 499

an improvement in insulin sensitivity associated with decrease in serum LH andandrogens In contrast, it has been shown91that the administration of metformin in

24 obese women, presenting with hirsutism according to the criteria of Ferrimanand Gallwey had no additional benefit over the effect of low caloric diet in improv-ing hyperinsulinaemia and hyperandrogenaemia Moreover, in this study therewas no control group for the weight loss intervention Recently, a study has been

published that supported the Vel´azquez et al results, in which administration of

metformin in obese women with PCOS reduces ovarian cytochrome P450c17activity and ameliorates hyperandrogenism and hirsutism by decreasing insulinconcentrations In these women the exaggerated serum 17α-hydroxyprogesteroneresponse to stimulation by gonadotropin-releasing hormone agonist was reducedafter metformin treatment.92

On the other hand, contradictory results were obtained in a study with a limitednumber of PCOS women with moderate to extreme obesity The study concludedthat hyperinsulinaemia and androgen excess in obese non-diabetic women withPCOS were not improved by the administration of metformin.93

Subsequently, one further study has been published to assess menstrual ity in 40 oligoamenorrheic women with PCOS After a six-month course ofmetformin an improvement in menstrual cyclicity and fertility was seen.94

cyclic-Another aspect that could be modified by metformin is the ovulatory response

to clomiphene The frequency of spontaneous ovulation and ovulation induced byclomiphene can be increased in obese women with PCOS by decreasing seruminsulin concentration with metformin.95 An improvement in menstrual patternafter metformin treatment has also been described and confirmed that, by reducinghyperinsulinism, metformin determines a reduction in intraovarian androgens.96This leads to a reduction in oestradiol levels and favours orderly follicular growth

in response to exogenous gonadotropins.97

In PCOS women with abdominal obesity, long-term treatment induced tion in body mass index associated with a significant improvement of hirsutismand menses abnormalities.98 Moreover, the 17-hydroxyprogesterone response tohuman chorionic gonadotropin was lower after metformin treatment,99 giving adirect demonstration that metformin leads to a reduction in stimulated ovariancytochrome P450c17 activity, concomitantly with a reduction in basal insulin andtestosterone levels and a significant increase in SHBG and IGFBP1.100

reduc-In conclusion, metformin reduced hyperinsulinaemia and hyperandrogenaemia,independently of changes in body weight In a large number of subjects thesechanges were associated with striking sustained improvements in menstrual abnor-malities and resumption of ovulation.101

Although not reported by all investigators, metformin seems to cause a decline

in insulin levels and reverses metabolic and ovarian abnormalities Many of thesechanges occur even in the absence of changes in body mass index

The action of metformin is not fully known It inhibits hepatic glucose

production and increases peripheral tissue sensitivity to insulin In vitro

therapeutic concentrations of metformin have been shown to stimulate the tyrosinekinase activity of the intracellular portion of theβ-subunit of the human insulinreceptors.102

We reported the results of 10-month treatment with metformin (850 mg twicedaily) in a lean girl aged 13 years and 6 months with severe hirsutism, acne, clitoralhypertrophy, acanthosis nigricans and primary amenorrhea Hormonal assessment

0 40 80 120

NS NS

0 10 20

*** **

0 50 100 150

*** ***

0 50 100

***

***

0 10

***

**

0 25 50 75

Figure 16.4 Clinical, endocrine and metabolic values before metformin treatment ( −) after

6 months of treatment (+) and 3 months after treatment (−) in adolescent girls with hirsutism, hyperandrogenism, oligomenorrhea, dyslipidaemia and hyperinsulinism after precocious pub- arche The top panel displays fasting glucose and mean serum insulin (MSI) during OGTT The middle panel shows changes in hirsutism score and FAI The bottom panel shows changes

in serum LDL and HDL cholesterol (J Clin Endocrinol Metab 2000, 85, 3526 – 3530, with

permission)

TREATMENT APPROACH WITH INSULIN SENSITIZERS 501

showed a severe insulin resistance with hyperinsulinaemia and naemia Molecular analysis of the insulin receptor gene showed a heterozygousmissense mutation (Val 1028) in exon 17 of the insulin receptor, abolishingautophosphorylation of the insulin receptorβ-subunit Basal androgens and fastinginsulin concentrations decreased significantly during treatment, whereas SHBGconcentration increased Breast development progressed and menarche occurred

hyperandroge-in the fifth month of therapy No side-effects were documented.103

The results commented on above encourage the use of metformin in naemic and hyperandrogenic women, but at present few studies have addressed theuse of metformin in children as a treatment for either insulin resistance PCOS.104

hyperinsuli-In non-obese adolescent girls with hirsutism, hyperinsulinism, hyperandrogenismand dyslipidaemia, metformin therapy tends to normalize these abnormalities inconcert.105Thus, in non-obese girls with an adolescent variant of PCOS, insulin-sensitizing treatment reduces hyperinsulinism, dyslipidaemia and hyperandro-genism and restores eumenorrhea and also induced ovulation106(Figure 16.4)

In conclusion, metformin was found to be an effective approach to reversemetabolic and ovarian abnormalities even in adolescent girls Prolonged treatmentwith metformin has been proved to be safe in type 2 diabetes mellitus and in apregnant hyperandrogenic woman.107 The most common morbidity associatedwith its use is gastrointestinal distress, specifically diarrhoea and abdominal pain,which is often transient and seems to be lessened if the dose is gradually increased

16.9 Treatment approach with insulin sensitizers

(thiazolidinediones)

Another class of insulin-sensitizing agents, the thiazolidinediones, have been used

to improve PCOS abnormalities These drugs require the presence of insulin, butthey do not stimulate insulin secretion They mainly activate a nuclear receptorcalled PPARγ (peroxisome proliferator-activated receptor gamma), which is moststrongly expressed in adipose tissue Activated PPARγ increases transcription

of certain insulin-sensitive genes, including those that code for GLUT4 glucosetransporters and enzymes for lipogenesis

The first thioglitazonedione, troglitazone, was introduced in Japan and the USA

in 1997 and withdrawn in 2000 due to reports of fatal idiosyncratic city.108 Other thiazolidinediones such as rosiglitazone and pioglitazone havelittle evidence of hepatotoxicity, except two non-fatal cases of hepatocellulardamage observed with the initiation of rosiglitazone therapy Thus, monitoring

hepatotoxi-of serum alanine transaminase should be performed before starting and duringtherapy.109, 110

Dunaif et al.111 evaluated 21 PCOS subjects who received either 200 or

400 mg/day troglitazone for 12 weeks in a randomized, double-blind study.Treatment with troglitazone resulted in significant improvement in insulin action.Increases in insulin sensitivity were significant at both doses of troglitazone

but were more marked at 400 mg than at 200 mg This was accompanied bydecreases in circulating insulin levels, both basally and after glucose load,which were accounted for almost entirely by changes at 400 mg troglitazonedose In this report, insulin sensitivity was improved independent of weightloss and hyperandrogenism was ameliorated The author claimed111 that thisobservation is consistent with the hypothesis that hyperinsulinaemia contributes tohyperandrogenism in PCOS However, the apparent dose-related effect suggeststhat these changes were troglitazone mediated.

Recently it could also be demonstrated that troglitazone improves the ovulatorydysfunction, hirsutism, hyperandrogenemia and insulin resistance of PCOS in adose-related fashion, with a minimum of adverse effects.112It should be noted thatnone of the insulin-sensitizing drugs have Food and Drug Administration (FDA)approval for use in PCOS, hirsutism or hyperandrogenism with insulin resistance.Considering that women with PCOS may have insulin resistance secondary

to a deficiency of D-chiro-inositol-containing phosphoglycans that mediateinsulin action, the administration of this substance could improve insulinsensitivity According to this hypothesis D-chiro-inositol increased insulin action

in patients with PCOS, thereby improving ovulatory function and decreasingserum androgen concentrations, hirsutism, blood pressure and plasma triglycerideconcentrations.113

The aforementioned results using insulin sensitizers give grounds for ing them a therapeutic approach for PCOS, alone or combined with antiandrogenicdrugs or oral contraceptives

The therapeutical interventions with insulin-sensitizing agents corroborate the ideathat insulin resistance with hyperinsulinaemia may indeed be a prime factor under-pinning the metabolic and hormonal disorders affecting anovulatory and ovarianhyperandrogenic women and adolescents

Randomized, controlled trials with safe insulin sensitizers will have to be ducted, especially in young women and adolescents with hyperinsulinism andanovulatory hyperandrogenism in an attempt to normalize insulin sensitivity andovarian function

con-Considering that insulin sensitizers have less effect on hirsutism than drogens, these drugs could be combined with an antiandrogen such as flutamide atlow doses Again, collaborative randomized trials in wide populations should beconducted to assess treatment results of the clinical and metabolic abnormalities

antian-in women and adolescents

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