Metabolic Lessons from the Study of Young Adolescents with Polycystic Ovary Syndrome—Is Insulin, Indeed, the Culprit?

Sharon E. Oberfield

Department of Pediatric Endocrinology Columbia University New York, New York 10032

Address correspondence and requests for reprints to: Sharon E. Oberfield, M.D., Professor of Pediatrics, Department of Pediatric Endocrinology, Columbia University, 630 West 168th Street, PH-5 East Room 522, New York, New York 10032.


    Introduction
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 Introduction
 References
 
The metabolic disturbances in type 2 diabetes include both peripheral insulin resistance and impaired insulin secretion (1). Polycystic ovary syndrome (PCOS), a metabolic condition observed in ~10% of reproductive age women, is characterized by hyperandrogenism and chronic anovulation. It is often associated with obesity, dyslipidemia, and acanthosis nigricans (AN). Insulin resistance is a common comorbidity in women with PCOS and is associated with increased risk for hypertension and cardiovascular disease. Studies have shown that 25–35% of obese women with PCOS, by 30 yr of age, will have either impaired glucose tolerance or type 2 diabetes (2). It has been suggested that PCOS should be considered as another metabolic abnormality of "Syndrome X," which includes dyslipidemia, hypertension, and insulin resistance (3).

The actual role of insulin (hyperinsulinemic insulin resistance) in the development and maintenance of hyperandrogenism in PCOS is a controversial area in which consensus has not been reached. In vitro and in vivo studies have shown that insulin acts at multiple sites, often leading to increased androgen excess and effect. Additionally, in women with PCOS, genetic factors, which may modulate the response to insulin at the cellular level, are just beginning to be described (for reviews see Refs. 4 and 5). An increase in serine/threonine phosphorylation of P450c17 increases the enzyme’s 17,20 lyase activity, resulting in an increase in androgen production, as seen in adrenarche (6). Increased serine phosphorylation of the insulin receptor ß chain causes insulin resistance by inhibiting tyrosine phosphorylation (7). The contemporaneous reports of Miller suggesting and Dunaif demonstrating that some women with PCOS have hyperphosphorylated insulin receptors allowed for speculation of the existence of a common molecular pathway—hyperactivity of a single serine kinase—for the two major features of PCOS, namely hyperandrogenism and insulin resistance (6, 7, 8). Recently, however, this was not confirmed in analysis of 17,20 lyase activity in cultured fibroblasts obtained from women with PCOS (9).

Insulin has been shown to directly stimulate ovarian androgen secretion through effects on steroidogenic enzymes (10). Nestler et al. (11, 12) have shown that these effects were partially reversed with use of metformin. It has been suggested that insulin also augments adrenal androgen synthesis (13). In vitro studies have demonstrated that insulin may also stimulate LH release directly or enhance LH release from pituicytes, thereby indirectly stimulating ovarian androgen production (14). Insulin may increase ovarian LH receptors (15). Insulin is known to decrease hepatic sex hormone-binding globulin (SHBG) production, allowing for an increase in the levels of circulating free androgens (16). Additionally, insulin has been shown in vitro to decrease insulin-like growth factor-binding protein I (IGFBP-I), which results in an increase in IGF-I, which may then act locally at the ovary to stimulate androgen synthesis (17). Thus, reduction of serum insulin would be expected to decrease some of the androgen-related symptomatology such as hirsutism. Decrease in insulin itself should also result in a decrease or modification in risk factors associated with hyperinsulinism and insulin resistance, including the development of type 2 diabetes, dyslipidemias, hypertension, and cardiovascular disease.

Most recently, insulin resistance has been demonstrated in girls with premature adrenarche (PA) (18, 19). PA, defined by the onset of pubic hair in girls before age 8 yr, is associated with adrenal androgen levels higher than expected for age. It has been suggested that PA may be an antecedent of PCOS (18, 20, 21). Indeed, although PA was once considered a benign condition, it is now known to be associated with risk for many of the same conditions as PCOS, including hirsutism, obesity, AN, hyperinsulinism, and insulin resistance (22, 23, 24). Of relevance is the hypothesis proposed by Barker et al. (25) that lower birth weight or reduced fetal growth is related to the development of type 2 diabetes mellitus, hypertension, and hyperlipidemia, as well as "Syndrome X" in adult life (26). Ibanez et al. (27, 28), a major proponent of an extension of this theory, state in this issue of the journal that a "constellation of hirsutism, hyperandrogenism, oligomenorrhea, dyslipidemia and hyperinsulinism in lean, young women may already be a late stage of a developmental disorder starting early in life" (29). Germane to the current report is the prior work by Apter et al. (30), in which hyperinsulinemic insulin resistance and reduced IGFBP-I and SHBG levels were associated with ovarian hyperandrogenism in young adolescent girls, suggesting that peripubertally insulin plays a contributory role to the development of the metabolic derangement ascribed to PCOS.

In the current report, Ibanez et al. (29) describe 10 "nonobese adolescents ... in whom the appearance of ovarian hyperandrogenism was heralded by premature pubarche, hyperinsulinism and dyslipidemia before puberty and, even earlier, by a low birth weight," in whom treatment with metformin, an insulin sensitizing agent, resulted in a decrease in hirsutism score, insulin response to an oral glucose tolerance test (OGTT), free androgen index, and androgen response to stimulation with GnRH. Serum triglyceride, total cholesterol, and LDL cholesterol levels declined whereas HDL cholesterol increased. Menses occurred within 4 months of treatment. Perhaps the most important aspect of this study is that metformin use was addressed in a young and lean population at risk for the development of cardiovascular disease. If these findings are reproduced in larger placebo and weight-controlled studies, significant implications could be drawn including the encouragement of the use of an insulin-sensitizing agent, like metformin, in young patients with hyperinsulinemia, particularly those with a family history of type 2 diabetes and cardiovascular disease.

At the present time, few studies have addressed the use of metformin in children as a treatment for either insulin resistance or obesity. The action of metformin is not fully known. It inhibits hepatic glucose production and increases peripheral tissue sensitivity to insulin. It is thought to decrease insulin action on both the ovary and the liver. In vitro, therapeutic concentrations of metformin have been shown to simulate the tyrosine kinase activity of the intracellular portion of the ß-subunit of the human insulin receptor, but higher levels of metformin inhibited the tyrosine kinases (31). The most common morbity associated with its use is gastrointestinal distress, specifically diarrhea and abdominal pain, which is often transient and seems to be lessened if the dose is gradually increased. Although patients with renal insufficiency seem to be most at risk of developing severe lactic acidosis after receiving metformin, a recent case was observed in an elderly patient with normal renal function (32).

Table 1 cites a limited number of recent studies that have assessed the efficacy of metformin as an agent capable of reversing metabolic and ovarian abnormalities often associated with increased levels of insulin.

Although not reported by all investigators, metformin seems to cause a decline in insulin levels, an increase in insulin sensitivity, and a decrease in LH, androgens, and lipid levels and the ratio of IGF-I/IGFBP-I. Many of these changes occur even in the absence of changes in body mass index (BMI). It may not been effective in profoundly obese women. It has been associated with modest declines in weight and increases in normal menstrual cycling patterns and ovulation. It has been used during pregnancy with safety. As seen in Table 1, use of metformin in adolescents has been extremely limited although its use seems to be safe in small pilot studies.

Comparison of the current study by Ibanez et al. (29) with the reports above suggests that the dose (1275 mg/day) and length of time (6 months) of metformin used is similar to the previous studies. The young mean age of the girls was 16.8 yr (range, 13–20; most were 15–17 yr, with one only 20 yr of age; personal communication) makes this of particular interest because efforts aimed at preventing long-term cardiovascular complications of hyperinsulinism and modifying effects of androgen excess in at-risk adolescents and young women of child-bearing age may be speculated to be more effective if begun at a younger age. Noteworthy is the fact that these adolescents were all at least 3 yr postmenarche and had been documented to have hyperinsulinemia—8 of 10 prepubertally at diagnosis of premature pubarche—and in early puberty, with mean serum insulin levels 2 or more SD scores above the mean for age. Thus, identification of an at-risk population would provide a younger-aged population in whom potential benefit from targeted therapy with insulin-sensitizing agents like metformin could be achieved.

Unlike the majority of women in the prior studies where metformin has been effective, these adolescents were lean (mean BMI, 21.9 kg/m2) and not obese. Other efforts aimed at reducing insulin levels in the Ibanez et al. (29) cohort, such as weight reduction, were, thus, not a real option. The significant decrease in hirsutism is noteworthy because other agents such as birth control pills or antiandrogens, which have been used to reduce the effects of hyperandrogenism in women with hyperinsulism and PCOS, have often resulted in limited success (52, 53). Although the skin findings of AN are frequently observed in obese women with insulin resistance and obesity, only one of these adolescents had mild AN (personal communication), perhaps because of their lean BMI. However PAI-1, another marker known to be associated with increased cardiovascular vascular events and insulin-resistance in obese and type 2 diabetic patients, was elevated and subsequently decreased with metformin use (54). Furthermore, although her population was not small for gestational age (i.e. weight less <10%), the mean birth weights were in the lowest quartile for gestational age (55), corroborating the previous reports linking low birth weight to insulin resistance in adulthood (25, 27).

As in many small initial clinical drug studies, there was no placebo control group; however, the reversal of improvements on withdrawal of metformin was impressive. Indeed, although this report is of a very small group of patients and may, in fact, represent a subgroup of girls at risk because of low birth weight, it supports the hypothesis that hyperinsulinemia may have an early critical role in the development of PCOS and "Syndrome X" and challenges us to confirm and expand these findings. Clearly, targeted treatment of a younger population of girls at risk for cardiovascular disease and type 2 diabetes associated with hyperinsulinism such as precocious pubarche may attenuate, decrease, or even prevent progression to PCOS in the reproductive years.

The public health ramification of this report may be quite significant. Currently, the incidence of type 2 diabetes in adolescents is increasing at an alarming rate (56). In Tokyo, Japan, for example, type 2 diabetes accounts for close to 80% of all childhood diabetes [Dr. K. Kida (Ehime University, Matsuyama, Japan), personal communication]. Is there sufficient safety and efficacy data available for metformin to suggest it be used in young patients at-risk for the development of type 2 diabetes?

As with any new therapeutic model, long-term safety issues need to be addressed. In the current study, minimal transient gastrointestinal discomfort was noted in three of the adolescents with no report of hypoglycemia or lactic acidosis. Use of insulin sensitizers must be carefully monitored as most recently evidenced by the Food and Drug Administration withdrawal of the thiazolidinedione troglitazone due to fatal liver toxicity associated with its use. At the present time, given the lack of established long-term efficacy and safety in either adults, adolescents, and certainly children, until prospective large-scale clinical trials are performed, it would seem prudent to limit the use of metformin to adolescents at risk for the development of PCOS and type 2 diabetes to clinical trials. The report by Ibanez et al. (29) should encourage initiation of such studies.




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Figure 1.
 
Received August 10, 2000.

Accepted August 13, 2000.


    References
 Top
 Introduction
 References
 

  1. Kahn CR, Vincent D, Doria A. 1996 Genetics of non-insulin dependent (type II) diabetes mellitus. Ann Rev Med. 47:509–531.[CrossRef][Medline]
  2. Ehrmann DA. 1997 Obesity and glucose intolerance in androgen excess. In: Azziz R, Nestler JE, Dewailly D, eds. Androgen excess disorders in women. Philadelphia: Lippincott-Raven; 705–712.
  3. Nestler JE. 1994 Assessment of insulin resistance. Sci Med. 1:58–67.
  4. Dunaif A. 1999 Insulin action in the polycystic ovary syndrome. Endocrinol Metab Clin North Am. 28:341–359.[Medline]
  5. Poretsky L, Cataldo NA, Rosenwaks Z, Giudice LC. 1999 The insulin-related ovarian regulatory system in health and disease. Endocr Rev. 20:535–582.[Abstract/Free Full Text]
  6. Zhang L, Rodriguez H, Ohno S, Miller WL. 1995 Serine phosphorylation of human P450c17 increases 17,20-lyase activity; implications for adrenarche and the polycystic ovary syndrome. Proc Natl Acad Sci USA. 92:10619–10623.[Abstract]
  7. Dunaif A, Xia J, Book C, Schenker E, Tang Z. 1995 Excessive insulin receptor serine phosphorylation in cultured fibroblasts and skeletal muscle. J Clin Invest. 96:801–810.[Medline]
  8. Auchus RJ, Geller DH, Lee TC, Miller WL. 1998 The regulation of human P450c17 activity: relationship to premature adrenarche, insulin resistance and the polycystic ovary syndrome. Trends Endocrinol Metab. 9:47–50.[CrossRef]
  9. Martens JW, Geller DH, Ossovskaya VS, et al. 17,20 Lyase activity in cultured fibroblasts stably expressing P450c17: no difference between patients with polycystic ovary syndrome and normal subjects. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 404 (Abstract).
  10. Rosenfield RL, Barnes RB, Cara JF, Uchy AW. 1990 Dysregulation of cytochrome P450c17{alpha} as a cause of polycystic ovarian syndrome. Fertil Steril. 53:785–791.[Medline]
  11. Nestler JE, Jakubowicz DJ. 1996 Decreases in ovarian cytochrome P450c17{alpha} activity and serum free testosterone after reduction of insulin secretion in polycystic ovary syndrome. N Engl J Med. 335:617–623.[Abstract/Free Full Text]
  12. Nestler JE, Jakubowicz DJ. 1997 Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17{alpha} activity and serum androgens. J Clin Endocrinol Metab. 82:4075–4079.[Abstract/Free Full Text]
  13. Moghetti P, Castello R, Negri C, et al. 1996 Insulin infusion amplifies 17{alpha}-hydroxycorticosteroid intermediates response to adrenocorticotropin in hyperandrogenic women: apparent relative impairment of 17,20-lyase activity. J Clin Endocrinol Metab. 81:881–886.[Abstract]
  14. Adashi EY, Hsueh AJ, Yen SS. 1981 Insulin enhancement of luteinizing hormone and follicle-stimulating release by cultured pituitary cells. Endocrinology. 108:1441–1449.[Abstract]
  15. Adashi EY, Resnick CE, D’Ercole AJ, Svoboda ME, vanWyk JJ. 1985 Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev. 6:400–420.[Abstract]
  16. Nestler JE, Powers LP, Matt DW, et al. 1991 A direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome. J Clin Endocrinol Metab. 72:83–89.[Abstract]
  17. Nobels F, Dewailly D. 1992 Puberty and polycystic ovarian syndrome; the insulin/insulin-like growth factor I hypothesis. Fertil Steril. 58:655–666.[Medline]
  18. DiMartino-Nardi J. 1998 Insulin resistance in prepubertal African-American and Hispanic girls with premature adrenarche: a risk factor for polycystic ovary syndrome. Trends Endocrinol Metab. 9:78–82.[CrossRef]
  19. Silfen ME, Manibo AM, Levine LS, Murphy AR, Oberfield SE. 2000 Fasting glucose to insulin ratio (FGIR) is a simple measure of insulin resistance in young girls with premature adrenarche (PA) or obesity. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 506 (Abstract).
  20. Vuguin P, Linder B, Rosenfeld RG, Saenger P, DiMartino-Nardi J. 1999 The roles of insulin sensitivity, insulin-like growth factor I (IGF-I), and IGF-binding protein-1 and -3 in the hyperandrogenism of African-American and Caribbean Hispanic girls with premature adrenarche. J Clin Endocrinol Metab. 84:2037–2042.[Abstract/Free Full Text]
  21. Ibanez L, Potau N, Virdis R, et al. 1993 Postpubertal outcome in girls diagnosed of premature pubarche during childhood: increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab. 76:1599–1603.[Abstract]
  22. Ibanez L, Potau N, Zampolli M, Rique S, Saenger P, Carrascosa A. 1997 Hyperinsulinemia and decreased insulin-like growth factor-binding protein-1 are common features in prepubertal and pubertal girls with a history of premature pubarche. J Clin Endocrinol Metab. 82:2283–2288.[Abstract/Free Full Text]
  23. Ibanez L, de Zegher F, Potau N. 1999 Anovulation after precocious pubarche: early markers and time course in adolescence. J Clin Endocrinol Metab. 84:2691–2695.[Abstract/Free Full Text]
  24. Ibanez L, Potau N, Chacon P, Pascual C, Carrascosa A. 1998 Hyperinsulinaemia, dyslipaemia and cardiovascular risk in girls with a history of premature pubarche. Diabetologia. 41:1057–1063.[CrossRef][Medline]
  25. Barker DJP, Hales CN, Fall CHD, Osmond C, Phipps K, Clark PMS. 1993 Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia. 36:62–67.[Medline]
  26. Jaquet D, Gaboriau A, Czernichow P, Levy-Marchal C. 2000 Insulin resistance early in adulthood in subjects born with intrauterine growth retardation. J Clin Endocrinol Metab. 85:1401–1406.[Abstract/Free Full Text]
  27. Ibanez L, Potau N, Francois I, de Zegher F. 1998 Precocious pubarche, hyperinsulinism, and ovarian hyperandrogenism in girls: relation to reduced fetal growth. J Clin Endocrinol Metab. 83:3558–3562.[Abstract/Free Full Text]
  28. Ibanez L, Potau N, Zegher F. 1999 Precocious pubarche, dyslipidemia, and low IGF binding protein-1 in girls: relation to reduced prenatal growth. Pediatr Res. 46:320–322.[Abstract]
  29. Ibanez L, Vall C, Potau N, Marcus MV, de Zegher F. 2000 Sensitization to insulin in adolescent girls to normalize hirsutism, hyperandrogenism, oligomenorrhea and hyperinsulinism after precocious pubarche. J Clin Endocrinol Metab. 85:0000–0000.
  30. Apter D, Bützow GA, Laughlin GA, Yen SSC. 1995 Metabolic features of polycystic ovary syndrome are found in adolescent girls with hyperandrogenism. J Clin Endocrinol Metab. 80:2966–2973.[Abstract]
  31. Stith BJ, Woronoff K, Wiernsperger N. 1998 Stimuation of the intracellular portion of the human receptor by the antidiabetic drug metformin. Biochem Pharmacol. 55:533–536.[CrossRef][Medline]
  32. Bhargava A, Iqbal Z, Kathula S, Premanandan J. 2000 Metformin associated lactic acidosis (MALA) in a patient with normal renal function. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 443 (Abstract).
  33. Velasquez EM, Mendoza S, Hamer T, Sosa F, Glueck CJ. 1994 Metformin therapy in polycystic ovary syndrome reduces hyperinsulinemia, insulin resistance, hyperandrogenemia, and systolic blood pressure, while facilitating normal menses and pregnancy. Metabolism. 43:647–654.[Medline]
  34. Acbay O, Bundogdu S. 1996 Can metformin reduces insulin resistance in polycystic ovary syndrome. Fertil Steril. 65:946–949.[Medline]
  35. Ehrmann DA, Cavaghan MK, Imperial J, Sturis J, Rosenfield RL, Polonsky KS. 1997 Effects of metformin on insulin secretion, insulin action, and ovarian steroidogenesis in women with polycystic ovary syndrome. J Clin Endocrinol Metab. 82:524–530.[Abstract/Free Full Text]
  36. Velazques E, Mendoza SG, Wang P, Glueck C. 1997 Metformin therapy is associated with a decrease in plasma plasminogen activator inhibitor-1, lipoprotein(a) and immunoreactive insulin levels in patients with the polycystic ovary syndrome. Metabolism. 46:454–457.[Medline]
  37. Morin-Papunen LC, Koivunen RM, Ruokonen A, Martikainen HK. 1998 Metformin therapy improves the menstrual pattern with minimal endocrine and metabolic effects in women with polycystic ovary syndrome. Fertil Steril. 69:691–696.[CrossRef][Medline]
  38. Unluhizarci K, Kelestimur F, Bayram F, Sahin Y, Tutus A. 1999 The effects of metformin on insulin resistance and ovarian steroidogenesis in women with polycystic ovary syndrome. Clin Endocrinol. 51:231–236.[CrossRef][Medline]
  39. DeLeo V, LaMarca A, Ditto A, Morgante G, Cianci A. 1999 Effects of metformin on gonadodtropin-induced ovulation in women with polycystic ovary syndrome. Fertil Steril. 72:282–285.[CrossRef][Medline]
  40. Glueck CJ, Wang P, Fontaine R, Tracy T, Sieve-Smith L. 1999 Metformin-induced resumption of normal menses in 39 of 43 (91%) previously amenorrheic women with the polycystic ovary syndrome. Metabolism. 48:511–519.[Medline]
  41. Pirwany IR, Yates RWS, Cameron IT, Fleming R. 1999 Effects of the insulin sensitizing drug metformin on ovarian function, follicular growth and ovulation rate in obese women with oligomenorrhoea. Hum Reprod. 14:1963–1968.
  42. LaMarca A, Morgante G, Paglia T, Ciotta L, Cianci A, DeLeo V. 1999 Effects of metformin on adrenal stroidogenesis in women with polycystic ovary syndrome. Fertil Steril. 72:985–989.[CrossRef][Medline]
  43. Sarlis NJ, Weil SJ, Nelson LM. 1999 Administration of metformin to a diabetic woman with extreme hyperandrogenemia of nontumoral origin: management of infertility and prevention of inadvertent masculinization of a female fetus. J Clin Endocrinol Metab. 84:1510–1513.[Free Full Text]
  44. DeLeo V, LaMarca A, Orvieto R, Morgante G. 2000 Effect of metformin on insulin-like growth factor (IGF) I and IGF-binding protein I in polycystic ovary syndrome. J Clin Endocrinol Metab. 85:1598–1600.[Abstract/Free Full Text]
  45. LaMarca A, Egbe TO, Morgante G, Paglia T, Ciana A, DeLeo V. 2000 Metformin treatment reduces cytochrome P-450c117{alpha} response to human chorionic gonadotrophin in women with insulin resistance-related polycystic ovary syndrome. Hum Reprod. 15:21–23.[Abstract/Free Full Text]
  46. Moghetti P, Castello R, Negri C, et al. 2000 Metformin effects on clinical features, endocrine and metabolic profiles, and insulin sensitivity in polycystic ovary syndrome: a randomized, double-blind, placebo controlled 6-month trial, followed by open, long-term clinical evaluation. J Clin Endocrinol Metab. 85:139–146.[Abstract/Free Full Text]
  47. Diamanti-Kandarakis E, Spina G, Kovli C, et al. Metformin treatment reduces endothelin-1 and androgen levels and improves insulin-resistance in women with polycystic ovary syndrome. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 565 (Abstract).
  48. Mogul H, Peterson SJ, Weinstein BI, Zhang S, Southren AL. Weight reduction and sustained weight loss with metformin and carbohydrate modified diet in the tereatment of nondiabetic women with midlife weight gain and hyperinsulinemia. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 500 (Abstract).
  49. Vandermolen DT, Ratts VS, Evans WS, Stovall DW, Kauma SW, Nestler SW. Metformin increases the ovulatory response to clomiphene citrate in patients resistant to CC alone. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 563 (Abstract).
  50. Haghi M, Jones KL, Gottschalk ME. Metformin improves menstrual regularity and hyperandrogenemia in adolescents with polycystic ovary syndrome. Proceedings of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000; p. 524 (Abstract).
  51. Freemark M, Bursey D. A therapeutic trial of metformin in obese adolescents predisposed to type 2 diabetes mellitus. Society for Pediatric Research, 2000; p. 128A (Abstract).
  52. Creatsas G, Koliopoulos C, Mastorakos G. 2000 Combined oral contraceptive treatment of adolescent girls with polycystic ovary syndrome. Ann NY Acad Sci. 900:245–252.[Abstract/Free Full Text]
  53. Diamanti-Kandarakis E, Zapanti E. 2000 Insulin sensitizers and antiandrogens in the treatment of polycystic ovary syndrome. Ann NY Acad Sci. 900:203–212.[Abstract/Free Full Text]
  54. Bavenholm P, Proudler A, Silveira A, et al. 1995 Relationships of insulin and intact and split proinsulin to haemostatic funstion in young men with and without coronary artery disease. Throm Haemost. 73:568–575.[Medline]
  55. Usher R, McLean F. 1969 Intrauterine growth of live-born Caucasian infants at sea level: standards obtained from measurements in 7 dimensions of infants born between 25 and 44 weeks of gestation. J Pediatr. 74:901–910.[Medline]
  56. American Diabetes Association. 2000 Type 2 diabetes in children and adolescents. Consensus statement. Diabetes Care. 22:381–389.