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Home , Aromatase deficiency, Hyperandrogenism

J Infertil Reprod Biol, 2016, Vol 4, No 1, pp: 1-5

Research Article

Relation between gene CYP19 variation and in Polycystic Syndrome Egyptian women

Rowaa A. Mostafa 1, Mohammed M. Al-Sherbeeny 1, Ibrahim A. Abdelazim 1,2*, Ahmed A. Fahmy 1, Mohamed M. Farghali 1, Mustafa A Abdel-Fatah 1, Manal Z.Mahran 3

1. Department of Obstetrics and Gynaecology, Ain-Shams University, Cairo, Egypt 2. Department of Obstetrics and Gynaecology, Ahmadi Hospital, Kuwait Oil Company (KOC), Kuwait 3. Department of Clinical Pathology, Ain-Shams University, Cairo, Egypt

Abstract Background: This study designed to detect the relation between the aromatase CYP19 gene SNP50 variation and hyperandrogenism in polycystic ovary syndrome (PCOS) Egyptian women. Methods: Sixty women included in this comparative study and divided into 2 groups; 30 PCOS women with clinical hyperandrogenism included in study group and 30 healthy non-PCOS women in control group. PCOS diagnosis based on the Rotterdam criteria. Studied women underwent complete physical examination with calculation of body mass index (BMI) and assessment of . Studied women subjected to hormonal profile and to CYP19 genotyping by collecting 5 ml whole blood on EDITA for DNA extraction and SNP 50 (rs2414096) genotype to detect the relation between the aromatase CYP19 gene SNP50 variation and hyperandrogenism in polycystic ovary syndrome Egyptian women. Results: Results showed that LH and LH/FSH ratio were significantly higher in PCOS women compared to controls. Also, serum and were significantly high in PCOS women compared to controls. CYP19 rs2424096 genotypic distribution AG alleles was significantly high in PCOS women compared to controls (11 (44%) versus 2 (8.4%); respectively) (p = 0.02). Estradiol/testosterone ratio was significantly low in AG genotypes PCOS compared to AG genotype controls (P = 0.001). Conclusion: In conclusion, CYP 19 rs2414096 polymorphism is associated with or reduced aromatase activity with subsequent hyperandrogenism in PCOS Egyptian women.

Keywords: Aromatase, Gene, Hyperandrogenism, PCOS, Egyptian women

1. Introduction Polycystic ovary syndrome (PCOS) is a complex step of by converting disorder affects 5%-6% of women during testosterone and to estradiol and reproductive age group (1). PCOS is typically estrone separately (8). PCOS has been observed in associated with menstrual irregularities, , women with aromatase deficiency or reduced hyperandrogenism, chronic and aromatase activity caused by rare loss-of function (2, 3). Ovarian overproduction mutations and antral follicles taken from PCOS is the key physio-pathologic feature of PCOS (4). women exhibited no aromatase activity (9-11). Women with PCOS and Aromatase deficiency or reduced aromatase have increased circulating testosterone activity in the ovarian follicles and the possible concentrations and genetic variation at the androgen excess resulting might contribute to , suggesting that hyperandrogen- abnormal follicle development seen in PCOS ism in both precocious puberty and PCOS may be women (12-15). The CYP19 gene is located on the partly genetically determined (5, 7). Aromatase long arm of chromosome 15 at position 15q21.1 (EC 1.14.14.1) is a member of the cytochrome and encodes aromatase (P450arom) (16). P450 family of enzymes (subfamily 19), which a It is reported that several single nucleotide key steroidogenic enzyme that catalyzes the final polymorphisms (SNPs) of the CYP19 gene are

*Corresponding address: Professor Ibrahim A. Abdelazim, Department of Obstetrics and Gynecology, Ain Shams University, Cairo, Egypt and Ahmadi Kuwait Oil Company (KOC) Hospital, Ahmadi, Kuwait. E-mail: [email protected]

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J Infertil Reprod Biol, 2016, Vol 4, No 1, pp: 1-5

associated with variation in serum androgen each primer, 25µl short tandem repeat (STR), 10x concentrations among women, both within and buffer (STR 10x buffer, Promega, Madison, Wl, between racial/ethnic groups (17). Several studies USA). The PCR was performed in a PTC-100 (MJ have reported the association of the SNP rs2414096 Research Inc., Waltham, United States) in the CYP19 gene with hyperandrogenism (16- thermocycler as follows: 30 cycles consisting of 1 18). In view of the strong evidence implicating the minute of denaturation at 94°C, 1 minute of importance of CYP19 SNP rs2414096 in androgen annealing at 60 °C and 1 minute of extension at metabolic pathways, this study designed to detect 72°C. An initial denaturation step of 5 minutes at the relation between the aromatase CYP19 gene 94°C and a final extension of 10 minutes at 72°C SNP50 variation and hyperandrogenism in PCOS used. The DNA fragments separated by Egyptian women. electrophoresis on a 2% agarose gel and visualized by staining with ethidium bromide. The PCR 2. Patients and methods products of 189-bp were then digested with HSP92 Sixty women included in this comparative study, ΙΙ at 37 °C overnight. A single 189 bp band which conducted at Ain Shams University corresponds to the wild-type G homozygote; bands Maternity hospital from September 2008 to June of 189, 161, and 28 bp stand for the AG 2011 and divided into 2 groups; 30 PCOS women heterozygotes; and 161 and 28 bp for the A with clinical hyperandrogenism mainly hirsutism homozygote. included in study group and 30 healthy non-PCOS women in control group. Studied women included 3. Results in this study after informed consent and approval of Mean age, BMI and FSH were similar with no the study by local institute ethical committee. statistical difference between PCOS women and Women with endocrinal disorders (thyroid controls. LH and LH/FSH ratio were significantly dysfunction, Cushing syndrome and high in PCOS compared to controls, also, serum hyperprolactinemia) and women received oral testosterone and estradiol were significantly high in contraceptives pills, or PCOS women compared to controls (Table 1). inducing medications during last 6 months CYP19 rs2424096 genotypic distribution AG excluded from the study. PCOS diagnosis based on alleles was significantly high in PCOS women the Rotterdam criteria by at least 2 out of 3 of the compared to controls (p = 0.02) (Table 2). following criteria: oligo-or an-ovulation, clinical or biochemical hyperandrogenism and polycystic Table 1. Characteristics of PCOS women and controls on trans-vaginal ultrasound (TVS) (17). P value, Studied women underwent complete physical Significance PCOS Control Variables 95% examination with calculation of body mass index n=30 n= 30 (BMI) and assessment of hirsutism by modified Confidence interval Ferriman Gallway score with a score ≥ 8 is P = 0.9 (>0.05) Age (Years) considered abnormal. Studied women subjected to NS Mean ± SD 31.1 ± 4.9 28.5 ± 7.4 hormonal profile including; follicle stimulating (-0.6, 2.6, 5.8) Body Mass P = 0.6 (>0.05)

hormone (FSH), (LH), Index (Kg/m 2) NS 27.6 ± 4.5 26.2 ± 4.8 testosterone and estradiol followed by TVS to Mean ± SD (-0.9, 1.4, 3.8) P = 0.9 (>0.05) detect polycystic ovaries according to criteria of FSH (IU/l) NS Mean ± SD 8.3 ± 3.5 7.7 ± 5.6 Balen and associates if present (18, 19). (-1.8, 0.6, 2.9) Studied women subjected to CYP19 genotyping P = 0.005 by collecting 5 ml whole blood on EDITA which LH (IU/l) (<0.05) S Mean ± SD 16.7 ± 2.1 3.5 ± 1.3 (12.3, 13.2, sent to Genetic Center for DNA extraction and 14.1) P = 0.008 SNP 50 (rs2414096) genotype using primer LH/FSH Ratio (<0.05) S Mean ± SD 2.01 ± 3.3 0.45 ± 2.1 Polymerase Chain Reaction (PCR) – Restriction (0.16, 1.6, 2.9) Fragment Length Polymorphism (RFLP) (20). Testosterone P = 0.03 (<0.05)

(nMol/l) S Genomic DNA was isolated from human 3.2 ± 3.8 1.2 ± 2.7 Mean ± SD (0.33, 2, 3.7) leukocytes by using Chelex-100 as a medium P = 0.03 (<0.05) Estradiol S (Promega Corporation, Madison, USA). The (pMol/l) 245.5 ± 198 176.2 ± 142.3 (-18.1, 69, Mean ± SD sequences of the primers were 5´- 156.6) TCTGGAAACTTTTGGTTTGAGTG-3´ (forward FSH = Follicle stimulating hormone. LH= Luteinizing primer) and 5´-GATTTAGCTTAAGAGCCTTTT hormone. NS = Non Significant difference. CTTACA-3´ (reverse primer). PCR amplification PCOS = Polycystic ovary syndrome. S= Significant difference. was carried out in a total volume of 25 µl Student t test used for statistical analysis

containing 50 ng of genomic DNA, 6.25 pmol of

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J Infertil Reprod Biol, 2016, Vol 4, No 1, pp: 1-5

4. Discussion Table 2. Comparison between PCOS women and controls A limited number of studies have focused on regarding CYP19 mutation, alleles and genotype in women’s health, particularly at the Controls P value, PCOS group genetic level (18), this why this study designed Variables = 30 Significance = 30 women detect the relation between the aromatase CYP19 Women gene SNP50 variation and the features of 0.8 (>0.05) Negative 5 (16.7%) 6 (20%) CYP19 NS hyperandrogenism in PCOS Egyptian women. mutation 1 (>0.05) Positive 25 (83.3%) 24 (80%) Sixty women included in this comparative study NS 11 0.6 (>0.05) and divided into 2 groups; 30 PCOS women with AA 9 (36%) (45.8%) NS clinical hyperandrogenism included in study group 0.02 (<0.05) Genotype AG 11 (44%) 2 (8.4%) and 30 healthy non-PCOS women in control group. S 11 0.18 (>0.05) PCOS diagnosis based on the Rotterdam criteria GG 5 (20%) (45.8%) NS (19). Studied women underwent complete physical Chi-Square test (x 2) used for statistical analysis. NS= Non examination with calculation BMI and assessment Significant difference. S = Significant difference . of hirsutism. Studied women subjected to hormonal profile and to CYP19 genotyping by collecting 5 Mean age, BMI and FSH were similar with no ml whole blood on EDITA for DNA extraction and significant difference between AG genotypes SNP 50 (rs2414096) genotype using primer PCR – Restriction Fragment Length Polymorphism PCOS and AG genotype controls. In addition, LH, LH/FSH ratio, testosterone and estradiol were (RFLP) (20). In this study, LH and LH/FSH ratio similar with no significant difference between AG were significantly high in PCOS Egyptian women genotypes PCOS and AG genotype controls. compared to controls; also, serum testosterone and estradiol were significantly high in PCOS Egyptian Estradiol/testosterone ratio was significantly low in AG genotypes PCOS compared to AG genotype women compared to controls. (Table 3). Ibrahim et al. and Dumesic et al. concluded that PCOS is a heterogeneous syndrome characterized by luteinizing hormone hypersecretion, ovarian Table 3. Comparison between Characteristics of AG hyperandrogenism and from genotypes PCOS women and controls resistance (1, 2). In this study, CYP19 Variables AG AG P value, rs2424096 genotypic distribution AG alleles was genotypes genotype Significance PCOS controls 95% CI significantly high in PCOS Egyptian women compared to controls and estradiol/testosterone Age (Years) P = 0.4 (>0.05) NS 28.5 ± 8.2 29.3 ± 6.3 ratio was significantly low in AG genotypes PCOS Mean ± SD (-10.7, 0.8, 9.18) compared to AG genotype controls. Petry and Body Mass colleagues suggested that common variation at the Index P = 0.5 (>0.05) NS 28.2 ± 7.4 27.5 ± 5.5 (Kg/m 2) (-8.08, 0.7, 9.48) aromatase gene (and not just rare loss-of-function Mean ± SD mutations) is associated with androgen excess in FSH (IU/l) P = 0.5 (>0.05) NS PCOS girls and young women (20). In addition, 7.6 ± 5.3 6.8 ± 3.9 Mean ± SD (-5.44, 0.8, 7.04) Guo et al. suggested the important effect of CYP19 on variation in age at (AAM) in LH (IU/l) P = 0.9 (>0.05) NS 18.9 ± 7.5 4.7 ± 2.4 Mean ± SD (8.18, 14.2, 20.2) Caucasian females (16). A large case controlled study including 684 individuals (386 PCOS women LH/FSH P = 0.8 (>0.05) NS and 298 controls) was performed to assess the Ratio 2.48 ± 4.4 0.7 ± 1.2 (-1.31, 1.78, 4.87) Mean ± SD association of SNP rs2414096 in the CYP 19 gene Testosterone in Chinese women by Jin and colleagues and they P = 0.8 (>0.05) NS (nMol/l) 2.9 ± 6.4 1.09 ± 0.8 (-2.13, 1.81, 5.75) found The genotypic distributions of rs2414096 Mean ± SD (GG, AG, AA) in the CYP19 gene (GG, AG, AA) Estradiol P = 0.7 (>0.05) NS 234.3 ± in women with PCOS (0.363, 0.474, 0.163, (pMol/l) 177.6 ± 186 (-211.29, 56.7, 124 Mean ± SD 324.7) respectively) were significantly different from that Estradiol/Tes P = 0.001 (<0.05) S in controls (0.242, 0.500, 0.258, respectively) (17). tosterone 80.8 ± 7.3 162.4 ± 6.8 (-91.96, -81.6, - Jin and colleagues, suggest that SNP rs2414096 in Ratio 71.24) Mean ± SD the CYP19 gene is associated with susceptibility to FSH = Follicle stimulating hormone. LH = Luteinizing PCOS (17). Sowers and colleagues evaluated hormone. NS = Non Significant difference. testosterone and estradiol (E2) levels among PCOS = Polycystic ovary syndrome. S = Significant women in relation to 5 single nucleotide difference. Student t test used for statistical analysis. polymorphisms (SNPs) of the aromatase (CYP 19) gene, the cytochrome P450 enzyme that converts 3

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androgens to (18). They related 5 -menarche after precocious pubarche: relation to aromatase SNPs (CYP 19 rs2414096, CYP 19 prenatal growth. Pediatrics 2006; 117:117-21. rs936306, CYP 19 rs2446405, CYP 19 rs1008805, 4. Diamanti-Kandarakis E, Piperi C. Genetics of and CYP 19 rs749292) to serum androgen and polycystic ovary syndrome: searching for the estradiol markers in 1,538 participants of the Study way out of the labyrinth. Hum Reprod Update. of Women’s Health Across the Nation (SWAN), 2005;11:631-643. and they found three of 5 aromatase gene SNPs 5. Escobar-Morreale HF, Luque-Ramâirez M, San were associated with variation in serum androgen Millâan JL. The molecular-genetic basis of concentrations among women, both within and functional hyperandrogenism and the polycystic between racial/ethnic groups (18). Sowers and ovary syndrome. Endocr Rev 2005;26:251-82. colleagues concluded that aromatase genetic 6. De Zegher F, Ibáñez L. Prenatal growth restraint markers might be important in understanding the followed by catch-up of weight: a emerging associations reported between hyperinsulinemic pathway to polycystic ovary endogenous androgens and women’s health status syndrome. Fertil Steril 2006;86 Suppl 1:S4-5. (18). In this study, the estradiol (E2)/testosterone 7. Vink J, Sadrzadeh SM, Lambalk CB, Boomsma ratio was significantly low in AG genotypes PCOS DI. Heritability of polycystic ovary syndrome compared to AG genotype controls (80.8 ± 7.3 (PCOS) in a Dutch twin-family study. J Clin versus 162.4 ± 6.8). Chen and colleagues found Endocrinol Metab 2006;91:2100-104. that the E2/testosterone was significantly lower in 8. Xita N, Georgiou I, Lazaros L, et al. The PCOS than non-PCOS controls and concluded that synergistic effect of -binding aromatase deficiency or reduced aromatase activity globulin and aromatase genes on polycystic is the main cause behind androgen excess in PCOS ovary syndrome phenotype. Eur J Endocrinol. women (12). Zhang and colleagues concluded that 2008;158:861-5. inhibited aromatase activity is the cause of 9. Xita N, Georgiou I, Lazaros L, et al. The role of hyperandrogenism in PCOS women. This is first sex hormone-binding globulin and androgen study conducted on Egyptian women to detect the receptor gene variants in the development of relation between the aromatase CYP19 gene polycystic ovary syndrome. Hum Reprod SNP50 variation and the features of 2008;23:693-8. hyperandrogenism in PCOS Egyptian women. The 10. Yang F, Ruan YC, Yang YJ, et al. Follicular strength of this study is coming from proper hyperandrogenism downregulates aromatase in statistical analysis and comparative nature of the luteinized granulosa cells in polycystic ovary study. This study concluded that CYP 19 syndrome women. Reproduction 2015;150: 289- rs2414096 polymorphism is associated with 96. aromatase deficiency or reduced aromatase activity 11. Zhang CW, Zhang XL, Xia YJ, et al. with subsequent hyperandrogenism in PCOS Association between polymorphisms of the Egyptian women. CYP11A1 gene and polycystic ovary syndrome in Chinese women. Mol Biol Rep 2012;39:8379- Conflict of interest 85. The authors declare no conflict of interest. 12. Chen J, Shen S, Tan Y, et al. The correlation of aromatase activity and obesity in women with or Financial disclosure without polycystic ovary syndrome. J Ovarian Study was funded by authors themselves. Res 2015;8:11. 13. Wang H, Li Q, Wang T, Yang G, et al. A

common polymorphism in the human aromatase Acknowledgement gene alters the risk for polycystic ovary Authors are very grateful to women agreed to syndrome and modifies aromatase activity in participate in this study. vitro. Mol Hum Reprod 2011;17: 386-91. 14. Zhang XL, Zhang CW, Xu P, et al. SNP References rs2470152 in CYP19 is correlated to aromatase 1. Ibrahim AA, Walid FE. Metabolic syndrome activity in Chinese polycystic ovary syndrome among infertile women with polycystic ovary patients. Mol Med Rep 2012;5:245-9. syndrome. Asian Pac J Reprod 2015;4:44-8. 15. Medeiros SF, Barbosa JS, Yamamoto MM. 2. Dumesic DA, Abbott DH, Padmanabhan V, et al. Comparison of steroidogenic pathways among Polycystic ovary syndrome and its normoandrogenic and hyperandrogenic developmental origins. Rev Endocr Metab polycystic ovary syndrome patients and normal Disord 2007;8:127-41. cycling women. J Obstet Gynaecol Res 3. Ibáñez L, Jiménez R, de Zegher F. Early puberty 2015;41:254-63. 4

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16. Guo Y, Xiong DH, Yang TL, et al. multiracial/multiethnic, multisite study of Polymorphisms of estrogen-biosynthesis genes women at midlife. Am J Med 2006;119:23-30. CYP17 and CYP19 may influence age at 19. Sun J, Fan H, Che Y, et al. Association between menarche: a genetic association study in ACE gene I/D polymorphisms and Caucasian females. Hum Mol Gen hyperandrogenism in women with polycystic 2006;15:2401-8. ovary syndrome (PCOS) and controls. BMC 17. Jin JL, Sun J, Ge HJ, et al. Association between Med Genet 2009;10:64. CYP19 gene SNP rs2414096 polymorphism and 20. Petry CJ, Ong KK, Michelmore KF, et al. polycystic ovary syndrome in Chinese women. Association of aromatase (CYP19) gene BMC Med Genet 2009;10:139. variation with features of hyperandrogenism in 18. Sowers MR, Wilson AL, Kardia SR, et al. two populations of young women. Hum Reprod Aromatase gene (CYP19) polymorphisms and 2005;20:1837-43. endogenous androgen concentrations in a

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