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The M235T polymorphism of the angiotensinogen gene in women with polycystic ovary syndrome
The M235T polymorphism of the angiotensinogen gene in women with polycystic ovary syndrome To explore the relationship between variation in AGT M235T gene and the development of the polycystic ovary syndrome (PCOS) and its sequelae, in the present study we evaluated AGT polymorphism M235T in women with PCOS and in a control group. Moreover, to detect any relationship between AGT M235T variation and intermediate and quantitative traits relevant to the pathogenesis of cardiovascular disease and PCOS, we looked for genotype-dependent differences within the subjects with PCOS. (Fertil Steril威 2005;84: 1520 –1. ©2005 by American Society for Reproductive Medicine.)
Polycystic ovary syndrome (PCOS), one of the most common endocrinopathies among women of reproductive age (1), displays strong familial aggregation. The etiology is multifactorial, individual susceptibility being determined by the action of multiple genetic and environmental risk factors (2). The abnormal hormonal milieu characteristic of PCOS may predispose to several conditions linked to insulin resistance, hyperandrogenism, or both (3) and women with PCOS appear to form a subset of the insulin resistance syndrome consisting of insulin resistance, hypertension, and lipid abnormalities. Furthermore, the dysfunctions associated with the renin-angiotensin system may participate in the pathogenesis of hyperandrogenism (4, 5) and of long-term PCOS sequelae such as insulin resistance and atherosclerosis (6, 7). The association between a renin-angiotensin system (RAS) polymorphism, M235T AGT gene polymorphism (a methionine-to-threonine amino acid substitution), and hypertension (8, 9) and other cardiovascular risk factors (10) has been reported by several investigators, but there are no data about the association between this polymorphism and PCOS. Given the epidemiological and genetic overlap between hypertension, cardiovascular diseases, and PCOS, the M235T AGT gene may represent a candidate gene for a role in PCOS susceptibility. To explore the relationship between variation in AGT M235T gene and the development of PCOS and its sequelae we studied 95 women with PCOS and 64 controls. The research protocol was approved by the local ethic boards of the participating institutions. The diagnosis of PCOS was made according to the criteria presented at the 1990 National Institute of Health Conference on PCOS. Controls consisted on a group of 64 women aged 70 ⫾ 5 Received July 14, 2004; revised and accepted May 4, 2005. Reprints requests: Carla Scaroni, M.D., Endocrinolgy Unit, University of Padova, via Ospedale 105, 35100 Padova, Italy (FAX: 390-49657391; E-mail: [email protected]).
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years, range 61– 80 years, with no history of cardiovascular diseases and with no evidence of hypertension. Basal blood samples were obtained to evaluate testosterone (20 –90 ng/dL), androstenedione (0.2–3 ng/mL), and DHEAS (0.7– 3.9 g/mL) levels. The oral glucose tolerance test was performed for measurement of glucose and insulin levels. Insulin resistance index was calculated by homeostasis model assessment (HOMA-IR) according to the formula: Plasma glucose (in mmol/liter) ⫻ Insulin (in U/mL)/22.5 (11). Total cholesterol and triglycerides levels were determined. Hirsutism was assessed using the modified Ferriman and Gallwey score. Blood pressure and body mass index (BMI) were measured in all the cases. Determination of the AGT M235T genotype was done with the polymerase chain reaction (PCR) detection method. The primers used were forward 5=-CAGGGTGCTGTCCACACTGGACCCC-3= and reverse 5=-CCGTTTGTGCAGGGCCTGGCTCTCT-3=. The digested PCR products were separated and visualized under UV lighting. The lengths of the fragments separated were 165 bp, corresponding to the M235 allele (M), and 141 bp, corresponding to the digested T235 allele (T). Genotypes were expressed as MM for homozygous normal, MT for heterozygous, and TT for homozygous mutant. Statistical analysis was performed by the 2 test, using the SPSS 9.0 (SPSS, Inc., Chicago, IL) package. A P value less than .05 was considered statistically significant. The frequency of the T allele was higher in women with PCOS (0.42) if compared with that reported (12) in a large Italian population sample (0.36) and with the one found in our selected population (0.30; P⬍.05) of older women not affected with hypertension or other cardiovascular diseases. The frequency of the homozygous TT genotype was significantly higher in patients than in the control group (23.4% vs. 16.4%, P⬍.05). The effect of the T allele on clinical and hormonal measures was next analyzed (Table 1). No differences in BMI, androgen levels, and FG score were found among genotypes.
Fertility and Sterility姞 Vol. 84, No. 5, November 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.05.043
TABLE 1 Clinical and hormonal data in patients with different genotypes.
BMI kg/m2 Testosterone (ng/mL) Androstenedione (ng/mL) DHEA-S (g/mL) HOMA-IR Score FG Systolic BP (mm Hg) Diastolic BP (mm Hg) Total cholesterol (mmol/L) Triglycerides (mmol/L)
MT (N ⴝ 40)
TT (N ⴝ 22)
MM (N ⴝ 33)
26.2 ⫾ 6.2 0.63 ⫾ 0.3 3.24 ⫾ 2 2.4 ⫾ 1.6 3.21 ⫾ 2.2a 9.7 ⫾ 4.5 123 ⫾ 14.2 80 ⫾ 9.4 4.6 ⫾ 1.1 1.2 ⫾ 0.6
24.4 ⫾ 5.5 0.56 ⫾ 0.23 3.18 ⫾ 1.4 3.1 ⫾ 1.4 3.43 ⫾ 1.3a 8.9 ⫾ 5.3 123.6 ⫾ 7.4 78.8 ⫾ 9 5 ⫾ 1b 1.02 ⫾ 0.47
24.6 ⫾ 4.6 0.77 ⫾ 0.2 3.81 ⫾ 1.4 2.1 ⫾ 1.2 2.02 ⫾ 0.7 10.2 ⫾ 5.7 125 ⫾ 11.7 79 ⫾ 5 4.3 ⫾ 0.5 0.97 ⫾ 0.4
Note: Data are expressed as mean ⫾ SD. a P⬍.05 vs. MM. b P⬍.05 vs. MM and MT. Zulian. M235T polymorphism of AGT gene in PCOS. Fertil Steril 2005.
The subjects with PCOS with the T allele seem to be more insulin resistant, as evidenced by a significantly higher HOMA index. Higher levels of angiotensin II and of angiotensinogen, its precursor, which have been demonstrated in subjects carrying the T allele, would be responsible for the amplification of insulin resistance. This seems to be more common in subjects with PCOS, judging from the higher prevalence of this allelic variant in these women. Our study confirms that the homozygosity of AGT M235T mutation is associated with high levels of cholesterol with no regard to the young age of our patients but no association was found between genotype and other components of the lipid profile such as triglycerides and high-density lipoprotein (HDL) cholesterol. Furthermore, a lack of significant association between the M235T mutation and systolic or diastolic blood pressure was observed, not precluding the possibility that the M235T mutation could be a marker for hypertension also in women with PCOS in later life. Elisa Zulian, M.D.a Paola Sartorato, M.D.a Francesca Schiavi, Ph.D.a Paolo Moghetti, M.D.b Roberto Castello, M.D.b Franco Mantero, M.D.a Giuseppe Opocher, M.D.a Carla Scaroni, M.D.a a Division of Endocrinology, Department of Medical and Surgical Sciences, University of Padova, Padova; and b Division of Endocrinology and Metabolic Diseases, University of Verona, Verona, Italy
Fertility and Sterility姞
REFERENCES 1. Solomon CG. The epidemiology of polycystic ovary syndrome. Prevalence and associated disease risks. Endocrinol Metab Clin North Am 1999;28:247– 63. 2. Legro RS, Strauss JF. Molecular progress in infertility: polycystic ovary syndrome. Fertil Steril 2002;78:569 –76. 3. Sozen I, Arici A. Hyperinsulinism and its interaction with hyperandrogenism in polycystic ovary syndrome. Obstet Gynecol Surv 2000;55: 321– 8. 4. Uncu G, Sozer MC, Develioglu O, Cengiz C. The role of plasma renin activity in distinguishing patients with polycystic ovary syndrome (PCOS) from oligomenorrheic patients without PCOS. Gynecol Endocrinol 2002;16:447–52. 5. Jaatinen TA, Matinlauri I, Anttila L, Koskinen P, Erkkola R, Irjala K. Serum total renin is elevated in women with polycystic ovarian syndrome. Fertil Steril 1995;63:1000 – 4. 6. Legro RS. Polycystic ovary syndrome. Long-term sequelae and management. Minerva Ginecol 2002;54:97–114. 7. Macut D, Micic D, Cvijovic G, Sumarac M, Kendereski A, Zoric S, et al. Cardiovascular risk in adolescent and young adult obese females with polycystic ovary syndrome (PCOS). J Pediatr Endocrinol Metab 2001;14:1353–9. 8. Jeunemaitre X. Genetic polymorphisms in the renin-angiotensin system. Therapie 1998;53:271–7. 9. Corvol P, Soubrier F, Jeunemaitre X. Molecular genetics of the renin-angiotensin-aldosterone system in human hypertension. Pathol Biol (Paris) 1997;45:229 –39. 10. Wang JG, Staessen JA. Genetic polymorphisms in the renin-angiotensin system: relevance for susceptibility to cardiovascular disease. Eur J Pharmacol 2000;410:289 –302. 11. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC, et al. Homeostasis model assessment: insulin resistance and beta-cells function from fasting plasma glucose and insulin concentrations in men. Diabetologia 1985;28:412–9. 12. Mettimano M, Lanni A, Migneco A, Specchia ML, Romano-Spica V, Savi L. Angiotensin-related genes involved in essential hypertension: allelic distribution in an Italian population sample. Ital Heart J 2001;2:589 –93.
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Polycystic ovary syndrome (PCOS), one of the most common endocrinopathies among women of reproductive age (1), displays strong familial aggregation. The etiology is multifactorial, individual susceptibility being determined by the action of multiple genetic and environmental risk factors (2). The abnormal hormonal milieu characteristic of PCOS may predispose to several conditions linked to insulin resistance, hyperandrogenism, or both (3) and women with PCOS appear to form a subset of the insulin resistance syndrome consisting of insulin resistance, hypertension, and lipid abnormalities. Furthermore, the dysfunctions associated with the renin-angiotensin system may participate in the pathogenesis of hyperandrogenism (4, 5) and of long-term PCOS sequelae such as insulin resistance and atherosclerosis (6, 7). The association between a renin-angiotensin system (RAS) polymorphism, M235T AGT gene polymorphism (a methionine-to-threonine amino acid substitution), and hypertension (8, 9) and other cardiovascular risk factors (10) has been reported by several investigators, but there are no data about the association between this polymorphism and PCOS. Given the epidemiological and genetic overlap between hypertension, cardiovascular diseases, and PCOS, the M235T AGT gene may represent a candidate gene for a role in PCOS susceptibility. To explore the relationship between variation in AGT M235T gene and the development of PCOS and its sequelae we studied 95 women with PCOS and 64 controls. The research protocol was approved by the local ethic boards of the participating institutions. The diagnosis of PCOS was made according to the criteria presented at the 1990 National Institute of Health Conference on PCOS. Controls consisted on a group of 64 women aged 70 ⫾ 5 Received July 14, 2004; revised and accepted May 4, 2005. Reprints requests: Carla Scaroni, M.D., Endocrinolgy Unit, University of Padova, via Ospedale 105, 35100 Padova, Italy (FAX: 390-49657391; E-mail: [email protected]).
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years, range 61– 80 years, with no history of cardiovascular diseases and with no evidence of hypertension. Basal blood samples were obtained to evaluate testosterone (20 –90 ng/dL), androstenedione (0.2–3 ng/mL), and DHEAS (0.7– 3.9 g/mL) levels. The oral glucose tolerance test was performed for measurement of glucose and insulin levels. Insulin resistance index was calculated by homeostasis model assessment (HOMA-IR) according to the formula: Plasma glucose (in mmol/liter) ⫻ Insulin (in U/mL)/22.5 (11). Total cholesterol and triglycerides levels were determined. Hirsutism was assessed using the modified Ferriman and Gallwey score. Blood pressure and body mass index (BMI) were measured in all the cases. Determination of the AGT M235T genotype was done with the polymerase chain reaction (PCR) detection method. The primers used were forward 5=-CAGGGTGCTGTCCACACTGGACCCC-3= and reverse 5=-CCGTTTGTGCAGGGCCTGGCTCTCT-3=. The digested PCR products were separated and visualized under UV lighting. The lengths of the fragments separated were 165 bp, corresponding to the M235 allele (M), and 141 bp, corresponding to the digested T235 allele (T). Genotypes were expressed as MM for homozygous normal, MT for heterozygous, and TT for homozygous mutant. Statistical analysis was performed by the 2 test, using the SPSS 9.0 (SPSS, Inc., Chicago, IL) package. A P value less than .05 was considered statistically significant. The frequency of the T allele was higher in women with PCOS (0.42) if compared with that reported (12) in a large Italian population sample (0.36) and with the one found in our selected population (0.30; P⬍.05) of older women not affected with hypertension or other cardiovascular diseases. The frequency of the homozygous TT genotype was significantly higher in patients than in the control group (23.4% vs. 16.4%, P⬍.05). The effect of the T allele on clinical and hormonal measures was next analyzed (Table 1). No differences in BMI, androgen levels, and FG score were found among genotypes.
Fertility and Sterility姞 Vol. 84, No. 5, November 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.05.043
TABLE 1 Clinical and hormonal data in patients with different genotypes.
BMI kg/m2 Testosterone (ng/mL) Androstenedione (ng/mL) DHEA-S (g/mL) HOMA-IR Score FG Systolic BP (mm Hg) Diastolic BP (mm Hg) Total cholesterol (mmol/L) Triglycerides (mmol/L)
MT (N ⴝ 40)
TT (N ⴝ 22)
MM (N ⴝ 33)
26.2 ⫾ 6.2 0.63 ⫾ 0.3 3.24 ⫾ 2 2.4 ⫾ 1.6 3.21 ⫾ 2.2a 9.7 ⫾ 4.5 123 ⫾ 14.2 80 ⫾ 9.4 4.6 ⫾ 1.1 1.2 ⫾ 0.6
24.4 ⫾ 5.5 0.56 ⫾ 0.23 3.18 ⫾ 1.4 3.1 ⫾ 1.4 3.43 ⫾ 1.3a 8.9 ⫾ 5.3 123.6 ⫾ 7.4 78.8 ⫾ 9 5 ⫾ 1b 1.02 ⫾ 0.47
24.6 ⫾ 4.6 0.77 ⫾ 0.2 3.81 ⫾ 1.4 2.1 ⫾ 1.2 2.02 ⫾ 0.7 10.2 ⫾ 5.7 125 ⫾ 11.7 79 ⫾ 5 4.3 ⫾ 0.5 0.97 ⫾ 0.4
Note: Data are expressed as mean ⫾ SD. a P⬍.05 vs. MM. b P⬍.05 vs. MM and MT. Zulian. M235T polymorphism of AGT gene in PCOS. Fertil Steril 2005.
The subjects with PCOS with the T allele seem to be more insulin resistant, as evidenced by a significantly higher HOMA index. Higher levels of angiotensin II and of angiotensinogen, its precursor, which have been demonstrated in subjects carrying the T allele, would be responsible for the amplification of insulin resistance. This seems to be more common in subjects with PCOS, judging from the higher prevalence of this allelic variant in these women. Our study confirms that the homozygosity of AGT M235T mutation is associated with high levels of cholesterol with no regard to the young age of our patients but no association was found between genotype and other components of the lipid profile such as triglycerides and high-density lipoprotein (HDL) cholesterol. Furthermore, a lack of significant association between the M235T mutation and systolic or diastolic blood pressure was observed, not precluding the possibility that the M235T mutation could be a marker for hypertension also in women with PCOS in later life. Elisa Zulian, M.D.a Paola Sartorato, M.D.a Francesca Schiavi, Ph.D.a Paolo Moghetti, M.D.b Roberto Castello, M.D.b Franco Mantero, M.D.a Giuseppe Opocher, M.D.a Carla Scaroni, M.D.a a Division of Endocrinology, Department of Medical and Surgical Sciences, University of Padova, Padova; and b Division of Endocrinology and Metabolic Diseases, University of Verona, Verona, Italy
Fertility and Sterility姞
REFERENCES 1. Solomon CG. The epidemiology of polycystic ovary syndrome. Prevalence and associated disease risks. Endocrinol Metab Clin North Am 1999;28:247– 63. 2. Legro RS, Strauss JF. Molecular progress in infertility: polycystic ovary syndrome. Fertil Steril 2002;78:569 –76. 3. Sozen I, Arici A. Hyperinsulinism and its interaction with hyperandrogenism in polycystic ovary syndrome. Obstet Gynecol Surv 2000;55: 321– 8. 4. Uncu G, Sozer MC, Develioglu O, Cengiz C. The role of plasma renin activity in distinguishing patients with polycystic ovary syndrome (PCOS) from oligomenorrheic patients without PCOS. Gynecol Endocrinol 2002;16:447–52. 5. Jaatinen TA, Matinlauri I, Anttila L, Koskinen P, Erkkola R, Irjala K. Serum total renin is elevated in women with polycystic ovarian syndrome. Fertil Steril 1995;63:1000 – 4. 6. Legro RS. Polycystic ovary syndrome. Long-term sequelae and management. Minerva Ginecol 2002;54:97–114. 7. Macut D, Micic D, Cvijovic G, Sumarac M, Kendereski A, Zoric S, et al. Cardiovascular risk in adolescent and young adult obese females with polycystic ovary syndrome (PCOS). J Pediatr Endocrinol Metab 2001;14:1353–9. 8. Jeunemaitre X. Genetic polymorphisms in the renin-angiotensin system. Therapie 1998;53:271–7. 9. Corvol P, Soubrier F, Jeunemaitre X. Molecular genetics of the renin-angiotensin-aldosterone system in human hypertension. Pathol Biol (Paris) 1997;45:229 –39. 10. Wang JG, Staessen JA. Genetic polymorphisms in the renin-angiotensin system: relevance for susceptibility to cardiovascular disease. Eur J Pharmacol 2000;410:289 –302. 11. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC, et al. Homeostasis model assessment: insulin resistance and beta-cells function from fasting plasma glucose and insulin concentrations in men. Diabetologia 1985;28:412–9. 12. Mettimano M, Lanni A, Migneco A, Specchia ML, Romano-Spica V, Savi L. Angiotensin-related genes involved in essential hypertension: allelic distribution in an Italian population sample. Ital Heart J 2001;2:589 –93.
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