Original Article Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
Received: March 26, 2013 Accepted after revision: August 28, 2013 Published online: October 19, 2013
Promoter Methylation of CYP19A1 Gene in Chinese Polycystic Ovary Syndrome Patients Ying-Ying Yu a Cui-Xiang Sun a Yin-Kun Liu b Yan Li b Li Wang c Wei Zhang a a
Obstetrics and Gynecology Hospital and b Liver Cancer Institute, Zhongshan Hospital, Fudan University, and Department of Reproductive Medicine, International Peace Maternity and Child Health Hospital, Shanghai JiaoTong University, Shanghai, PR China
c
Abstract Aims: This study aimed to examine the methylation status of the CYP19A1 promoter region in Chinese polycystic ovary syndrome (PCOS) patients. Methods: A case-control study was designed that involved 10 PCOS patients and 10 controls. Ovary tissues obtained from 10 women with PCOS and 10 healthy controls were matched for body mass index and age. Methylation of CYP19A1 promoter was detected by methylation-specific PCR. CYP19A1 expression was measured by real-time PCR and Western blotting. Results: The methylation level of CYP19A1 promoter in PCOS samples was significantly higher than in controls (0.698 ± 0.192 vs. 0.210 ± 0.064, p < 0.01). A significant downregulation of CYP19A1 mRNA and protein expression levels was observed in PCOS ovary tissues. Furthermore, the scatter plot revealed that promoter methylation was inversely correlated with CYP19A1 mRNA level (Pearson’s correlation –0.820, p < 0.01). Conclusion: CYP19A1 expression is frequently repressed in PCOS ovaries due to the promoter hypermethylation. CYP19A1 promoter hypermethylation may play a key role in the pathogenesis of PCOS. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0378–7346/13/0764–0209$38.00/0 E-Mail [email protected] www.karger.com/goi
Introduction
Polycystic ovary syndrome (PCOS) is a common heterogeneous disorder in women of child-bearing age, and the most common cause of female infertility due to anovulation [1, 2]. Whilst the pathogenesis of PCOS remains largely unknown, accumulating data suggest that PCOS is a complex disease which involves both genetic and environmental factors [3–5]. PCOS may begin in utero, because female mammals (rhesus monkeys, sheep, mice or rats) exposed to androgen in utero develop masculinized phenotypes similar to PCOS in adulthood [6– 8]. Obesity in adolescents likely exacerbates the signs of this condition [9, 10]. Diet and exercise, such as a wellbalanced diet with restricted caloric intake and portion size, combined with regular and moderate physical activity, are recommended as the first-line treatment for PCOS by the majority of endocrinologists and gynecologists [11, 12]. Therefore, it is postulated that environmental factors may reprogram the genome, causing persistent DNA methylation modification and irregular gene expression predisposing to PCOS [13]. The cytochrome P450 family 19 subfamily A polypeptide 1 gene (CYP19A1) encodes aromatase (EC 1.14.14.1), a key steroidogenic enzyme that catalyzes the final step of Dr. Wei Zhang Obstetrics and Gynecology Hospital, Fudan University 413 Zhaozhou Road Shanghai 200011 (PR China) E-Mail weizhang 611 @ 163.com
Downloaded by: NYU Medical Center Library 128.122.253.228 - 5/14/2015 4:00:32 PM
Key Words Polycystic ovary syndrome · CYP19A1 · Methylation
Materials and Methods Patient Selection and Sample Collection Ten PCOS patients with 10 controls were recruited from the Obstetrical and Gynecological Hospital of Fudan University. All participants were of Chinese Han ethnicity. The study was approved by the institutional ethics committee and informed consent was obtained from all subjects participating in the study. PCOS patients were selected from those who underwent ovarian drilling for ovulation induction and were diagnosed based on the 2003 Rotterdam Criteria [18]. Controls were 10 normal menstruating women without signs of hyperandrogenism who underwent laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain or oophorectomy for nonovarian indications. Endocrine parameters in control subjects were all within the normal range. Ovarian tissue samples were collected from all participants, histologically examined and stored in liquid nitrogen. For each PCOS subject, a control subject was matched for age (±3 years) and body mass index (BMI; ±3) to statistically adjust for these two variables. Exclusion criteria for the study included hyperprolactinemia, thyroid disease, adrenal hyperplasia, Cushing’s syndrome, androgen-secreting tumors, endometriosis, known cardiovascular disease, neoplasms, diabetes, hypertension (blood pressure >140/90 mm Hg) and other conditions that may affect the results. None of the subjects had taken medications for at least 3 months before the study, including oral contraceptives, glucocorticoids, ovulation induction agents, antidiabetic and antiobesity drugs, or estrogenic, antiandrogenic or antihypertensive medication. Bisulfite Modification and Methylation-Specific PCR Bisulfite modification of 1 μg genomic DNA was performed by using an EZ DNA methylation kit (Zymo Research, Orange, Calif., USA) according to the manufacturer’s instructions and eluted into a 20-μl volume, which was then diluted to give a final concentration of 20 ng/μl. One microliter of bisulfite-treated genomic DNA was amplified using GC Rich DNA polymerase (Qiagen, Hilden, Germany) and the following primers: methylation-specific primers, forward 5′-GTTTAATTCGGTTAGGAAGGAGC-3′, reverse 5′-CGACTTACCCGAAACGTATAAAA-3′, 294 bp; unmethyl-
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Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
ation primers, forward 5′-GGTTTAATTTGGTTAGGAAGGA GT-3′, reverse 5′-ACAACTTACCCAAAACATATAAAA-3′, 296 bp. PCR was carried out using an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA) under the following conditions: 1 cycle of 95 ° C for 5 min, followed by 35 cycles of denaturing at 94 ° C for 45 s, annealing at 60 ° C for 45 s, extension at 72 ° C for 45 s and final extension at 72 ° C for 7 min. PCR products were analyzed by electrophoresis on 2% agarose gel. Lymphocyte DNA purified from peripheral blood of a healthy voluntary donor was used as the negative unmethylation control. In addition, the DNA was treated in vitro with SssI-CpGMethylase (NEB, Frankfurt, Germany) and used as a positive methylation control. The optical density values for each amplified product corresponded to the subtraction of background values from medium band intensity. The extent of methylation was semiquantitatively scored by the summation of: mean optic density of the methylated PCR product/(mean optic density of the methylated PCR product + mean optic density of the unmethylated PCR product), and the final score was defined as the methylation level.
Quantitative Reverse-Transcription PCR Total RNA from individual ovarian tissues or granulose cells was isolated using Trizol (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer’s instructions. Two micrograms of total RNA was used as a template for RT-PCR. Single-stranded complementary DNA was transcribed using a PrimeScript® RTPCR Kit (Perfect Real-Time, TAKARA, Dalian, China). Quantitative RT-PCR was performed using Fast Start DNA Master SYBR Green I kit (Roche, Mannheim, Germany) with the following primers: CYP19A1, forward 5′-GCTGGAAATGATCTTTACC CCA-3′ and reverse 5′-TGTAGCCTGGTTCTCTGGTGTG-3′; GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCA CCCTGTTGCTGTA-3′. Western Blot Analysis Human ovarian tissues were homogenized as described previously [19]. The homogenates were centrifuged at 12,000 g at 4 ° C for 20 min and the protein concentration in the supernatants was estimated using the Lowry method [20], as described before [19]. Bands were detected by using enhanced chemiluminescence assay and analyzed by densitometry using the Image Pro-Plus 3.0.1 system (Media Cybernetics, Silver Spring, Md., USA) with β-actin as the loading control.
Statistical Analysis Data were expressed as mean ± SD and analyzed using SPSS software version 16.0 (SPSS, Chicago, Ill., USA). Paired comparisons were performed using the paired, two-sided Student t test. The associations between variables were accessed using Pearson’s correlation test. p < 0.05 was considered as statistically significant.
Results
The General Characteristics of PCOS Patients and Controls The general characteristics of PCOS patients and controls are shown in table 1. No significant difference beYu/Sun/Liu/Li/Wang/Zhang
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estrogen biosynthesis by converting testosterone to estradiol [14]. The production of estrogen in the ovary is essential for follicular growth and selection [15]. Alterations in these processes could at least in part be responsible for the development of follicular cysts [16]. PCOS is observed in patients with aromatase deficiency due to rare loss-of-function mutations [17]. Therefore, CYP19A1 may act as a genetic modifier of the hyperandrogenic phenotype of PCOS. In this study, we speculated that the methylation of the CYP19A1 promoter region may be associated with decreased aromatase activity and hyperandrogenism in PCOS women. To test this hypothesis, we compared the methylation of CYP19A1 promoter and the expression level of CYP19A1 in PCOS patients and controls.
trol group
300 bp M
M.Sssl
U
M
Lymphocyte
U P1
1.00
0.604 0.924 0.588 0.012* 0.601 0.001* 0.870 0.024* 0.040* 0.020*
* p < 0.05.
tween age and BMI was seen between the two groups. Women with PCOS had higher levels of testosterone and luteinizing hormone (LH; p < 0.01) with higher LH/follicle-stimulating hormone (FSH) ratio (p < 0.05) and larger ovarian volume (p < 0.05) when compared to women without PCOS. The estrogen to androgen (E2/T) ratio was significantly lower in PCOS than that in control (p < 0.05). CYP19A1 Promoter Methylation Level in PCOS Patients and Controls Methylation-specific PCR (MSP) results showed that the promoters were methylated in all the investigated ovarian tissue samples (fig. 1). The relative methylation level of PCOS samples was 0.698 ± 0.192, while that of controls was 0.210 ± 0.064. A significant difference in the CYP19A1 promoter methylation level between PCOS and control groups was seen (p < 0.01). CYP19A1 mRNA Expression Level in PCOS Patients and Controls Real-time RT-PCR analysis showed that the CYP19A1 mRNA expression level was significantly lower in PCOS patients than in controls. The ratio of CYP19A1/GAPDH mRNA was 0.154 ± 0.012 in the PCOS group and 0.514 ± 0.132 in control subjects (p < 0.01; fig. 2).
M
U P2
M
U C1
M
U C2
p < 0.01
0.80
Marker
0.60 0.40 0.20 0
b
Control
PCOS
Fig. 1. MSP analysis of the methylation of CYP19A1 promoter in the two groups. a Representative gel image of MSP; MSP products: U represents the unmethylated product (296 bp); M represents the methylated product (294 bp); Lymphocyte represents the negative unmethylation control; M.SssI represents a positive methylation control; P1 and P2 represent two randomly selected samples in the PCOS group, and C1 and C2 represent two randomly selected samples in the control group. b The methylation of CYP19A1 promoter was semiquantitatively scored as the methylation level, which was 0.698 ± 0.192 for the PCOS group, significantly higher than 0.2098 ± 0.0642 in the control group (n = 10; p < 0.01).
p < 0.01
0.60
0.40
0.20
0
Control
PCOS
CYP19A1 Protein Expression Level in PCOS Patients and Controls Western blot analysis showed that the expression level of CYP19A1 protein was significantly lower in PCOS pa-
Fig. 2. Quantitative RT-PCR analysis of CYP19A1 mRNA expression in the two groups. The relative mRNA level of CYP19A1 was normalized to that of GAPDH. The ratio of CYP19A1/ GAPDH mRNA was 0.154 ± 0.012 in the PCOS group, significantly lower than 0.514 ± 0.132 in the control group (n = 10; p < 0.01).
CYP19A1 Expression Is Repressed by Promoter Hypermethylation in PCOS
Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
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10 29.4±3.7 22.5±3.0 6.6±2.9 5.2±2.0 47.7±22.8 0.4±0.1 11.7±5.5 0.9±0.5 136.0±75.1 7.2±2.8
a
M
Color version available online
10 28.4±4.7 22.7±3.4 7.2±1.4 14.9±10.2 43.0±13.0 0.7±0.20 12.2±7.4 2.1±1.4 71.4±44.4 13.7±5.3
p value
Relative mRNA level of CYP19A1
Controls
Relative methylation level of CYP19A1 promoter
n Age, years BMI, kg/m2 FSH, mIU/ml LH, mIU/ml E2, pg/ml Testosterone, ng/ml Prolactin, ng/ml LH/FSH E2/T Ovarian volume, cm3
PCOS
U
Color version available online
Table 1. General characteristics of women with PCOS and the con-
P3
P4
C1
C2
C3
a
S 0.15
0.10
0.40
0.20
0 0
&RQWURO
PCOS
Fig. 3. Western blot analysis of CYP19A1 protein expression in the two groups. a Representative blots for the detection of CYP19A1 protein level; β-actin was used as a loading control. P1, P2, P3 and P4 represent four randomly selected samples in the PCOS group; C1, C2, C3 and C4 represent four randomly selected samples in the control group. b Densitometric analysis of CYP19A1 protein level; the relative protein level of CYP19A1 was normalized to that of β-actin. The ratio of CYP19A1/β-actin protein was 0.037 ± 0.009 in the PCOS group, significantly lower than 0.134 ± 0.031 in control group (n = 10; p < 0.01).
tients than in the controls. The ratio of CYP19A1/β-actin protein level was 0.037 ± 0.009 in the PCOS group and 0.134 ± 0.031 in the control group (p < 0.01; fig. 3). Correlation between CYP19A1 Promoter Methylation and mRNA Expression Levels The scatter plot revealed that the level of methylation was inversely correlated with CYP19A1 mRNA level, and Pearson’s correlation coefficient was –0.820 (p < 0.01; fig. 4). The results showed a significant correlation between the promoter methylation and mRNA expression level of CYP19A1.
Discussion
Methylation modification of DNA is a mechanism by which the environmental factors affect the gene expression without altering the genetic code [21]. Emerging ev212
PCOS Control
0.60
0.05
0
b
Pearson’s correlation coefficient = –0.820 p < 0.01
Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
0.20
0.40
0.60
0.80
1.00
Relative methylation level of CYP19A1 promoter
Fig. 4. Scatter plot of CYP19A1 mRNA expression level versus pro-
moter methylation level in PCOS patients. The methylation level was inversely correlated with the CYP19A1 mRNA expression level. Pearson’s correlation coefficient was –0.820 (p < 0.01).
idence from prenatally androgenized models provides new insights into the pathogenesis of PCOS and suggests that environmental factors may predispose individuals to this disorder [4]. Moreover, the inactivation of the X chromosome and androgen receptor methylation associate with the manifestation of PCOS phenotypes [22, 23]. In this case study, we investigated the methylation of the CYP19A1 promoter in PCOS ovaries. Our results showed that the CYP19A1 promoter methylation level was significantly higher in PCOS samples than in controls. The E2/T ratio was used as an indirect measure for aromatase activity [24]. Compared with controls, PCOS had lower levels of E2/T (p < 0.05), and CYP19A1 mRNA and protein levels were both significantly lower in PCOS ovaries than in control samples. Moreover, Pearson’s correlation test showed that CYP19A1 gene promoter methylation level was inversely correlated with CYP19A1 mRNA level. Based on the present data we believe that reduced CYP19A1 expression in PCOS ovaries may be due to gene repression by DNA methylation of the CYP19A1 promoter. Ovarian androgen overproduction is a key pathologic feature of PCOS [25]. The enzyme aromatase encoded by the CYP19A1 gene mediates the conversion of the anYu/Sun/Liu/Li/Wang/Zhang
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5HODWLYHSURWHLQOHYHORICYP19A1
0.20
C4
Color version available online
P2
Relative mRNA level of CYP19A1
P1
Color version available online
CYP19A1 (58 kDa) DŽ$FWLQ (43 kDa)
drogens testosterone and androstenedione to the estrogens, E2, and estrone. Our findings suggest that methylation modification is a factor that affects aromatase expression and activity and thus influences estrogen biosynthesis. In conclusion, we demonstrated that the presence of methylation of the CYP19A1 promoter is higher in PCOS patients and correlates with the lower aromatase expression. These findings suggest that CYP19A1 expression is frequently repressed in PCOS ovaries due to
the promoter hypermethylation. CYP19A1 promoter hypermethylation may play a key role in the pathogenesis of PCOS. Acknowledgements We are extremely grateful to all the women who agreed to participate in our study. We thank Mr. Kun Guo of the Liver Cancer Institute, Zhongshan Hospital, Fudan University, for his helpful advice.
References
CYP19A1 Expression Is Repressed by Promoter Hypermethylation in PCOS
10 Pfeifer SM, Kives S: Polycystic ovary syndrome in the adolescent. Obstet Gynecol Clin North Am 2009;36:129–152. 11 Giallauria F, Palomba S, Maresca L, Vuolo L, Tafuri D, Lombardi G, Colao A, Vigorito C, Francesco O: Exercise training improves autonomic function and inflammatory pattern in women with polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf) 2008;69:792– 798. 12 Thomson RL, Buckley JD, Noakes M, Clifton PM, Norman RJ, Brinkworth GD: The effect of a hypocaloric diet with and without exercise training on body composition, cardiometabolic risk profile, and reproductive function in overweight and obese women with polycystic ovary syndrome. J Clin Endocrinol Metab 2008;93:3373–3380. 13 Xu N, Azziz R, Goodarzi MO: Epigenetics in polycystic ovary syndrome: a pilot study of global DNA methylation. Fertil Steril 2009;94: 781–783. 14 Petry CJ, Ong KK, Michelmore KF, Artigas S, Wingate DL, Balen AH, de Zegher F, Ibanez L, Dunger DB: Association of aromatase (CYP 19) gene variation with features of hyperandrogenism in two populations of young women. Hum Reprod 2005; 20: 1837– 1843. 15 Britt KL, Saunders PK, McPherson SJ, Misso ML, Simpson ER, Findlay JK: Estrogen actions on follicle formation and early follicle development. Biol Reprod 2004; 71: 1712– 1723. 16 Behera M, Price T, Walmer D: Estrogenic ovulatory dysfunction or functional female hyperandrogenism: an argument to discard the term polycystic ovary syndrome. Fertil Steril 2006;86:1292–1295. 17 Xita N, Lazaros L, Georgiou I, Tsatsoulis A: CYP19 gene: a genetic modifier of polycystic ovary syndrome phenotype. Fertil Steril 2010; 94:250–254.
18 Geisthovel F: A comment on the European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine consensus of the polycystic ovarian syndrome. Reprod Biomed Online 2003; 7: 602–605. 19 Chapdelaine P, Vignola K, Fortier MA: Protein estimation directly from SDS-PAGE loading buffer for standardization of samples from cell lysates or tissue homogenates before Western blot analysis. Biotechniques 2001;31: 478, 480, 482. 20 Gong XW, Wei DZ, He ML, Xiong YC: Lowry method for the determination of pegylated proteins: the error, its reason, and a method for eliminating it. Anal Biochem 2006; 354: 157–158. 21 Sharma A, Heuck CJ, Fazzari MJ, Mehta J, Singhal S, Greally JM, Verma A: DNA methylation alterations in multiple myeloma as a model for epigenetic changes in cancer. Wiley Interdiscip Rev Syst Biol Med 2010;2:654–669. 22 Dasgupta S, Sirisha PV, Neelaveni K, Anuradha K, Reddy AG, Thangaraj K, Reddy BM: Androgen receptor CAG repeat polymorphism and epigenetic influence among the south Indian women with polycystic ovary syndrome. PLoS One 2010;5:e12401. 23 Hickey TE, Legro RS, Norman RJ: Epigenetic modification of the X chromosome influences susceptibility to polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91:2789–2791. 24 Zhang XL, Zhang CW, Xu P, Liang FJ, Che YN, Xia YJ, Cao YX, Wu XK, Wang WJ, Yi L, Gao Q, Wang Y: SNP rs2470152 in CYP19 is correlated to aromatase activity in Chinese polycystic ovary syndrome patients. Mol Med Rep 2012;5:245–249. 25 Christakou CD, Diamanti-Kandarakis E: Role of androgen excess on metabolic aberrations and cardiovascular risk in women with polycystic ovary syndrome. Womens Health (Lond Engl) 2008;4:583–594.
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1 Aubuchon M, Legro RS: Polycystic ovary syndrome: current infertility management. Clin Obstet Gynecol 2011;54:675–684. 2 Gerli S, Casini ML, Unfer V, Costabile L, Mignosa M, Di Renzo GC: Ovulation induction with urinary FSH or recombinant FSH in polycystic ovary syndrome patients: a prospective randomized analysis of cost-effectiveness. Reprod Biomed Online 2004;9:494– 499. 3 Li C, Shi Y, You L, Wang L, Chen ZJ: Melatonin receptor 1A gene polymorphism associated with polycystic ovary syndrome. Gynecol Obstet Invest 2011;72:130–134. 4 Li Z, Huang H: Epigenetic abnormality: a possible mechanism underlying the fetal origin of polycystic ovary syndrome. Med Hypotheses 2008;70:638–642. 5 Maier PS, Spritzer PM: Aromatase gene polymorphism does not influence clinical phenotype and response to oral contraceptive pills in polycystic ovary syndrome women. Gynecol Obstet Invest 2012;74:136–142. 6 Bruns CM, Baum ST, Colman RJ, Dumesic DA, Eisner JR, Jensen MD, Whigham LD, Abbott DH: Prenatal androgen excess negatively impacts body fat distribution in a nonhuman primate model of polycystic ovary syndrome. Int J Obes (Lond) 2007; 31: 1579– 1585. 7 Dumesic DA, Abbott DH, Padmanabhan V: Polycystic ovary syndrome and its developmental origins. Rev Endocr Metab Disord 2007;8:127–141. 8 Abbott DH, Barnett DK, Levine JE, Padmanabhan V, Dumesic DA, Jacoris S, Tarantal AF: Endocrine antecedents of polycystic ovary syndrome in fetal and infant prenatally androgenized female rhesus monkeys. Biol Reprod 2008;79:154–163. 9 Franks S: Genetic and environmental origins of obesity relevant to reproduction. Reprod Biomed Online 2006;12:526–531.
Received: March 26, 2013 Accepted after revision: August 28, 2013 Published online: October 19, 2013
Promoter Methylation of CYP19A1 Gene in Chinese Polycystic Ovary Syndrome Patients Ying-Ying Yu a Cui-Xiang Sun a Yin-Kun Liu b Yan Li b Li Wang c Wei Zhang a a
Obstetrics and Gynecology Hospital and b Liver Cancer Institute, Zhongshan Hospital, Fudan University, and Department of Reproductive Medicine, International Peace Maternity and Child Health Hospital, Shanghai JiaoTong University, Shanghai, PR China
c
Abstract Aims: This study aimed to examine the methylation status of the CYP19A1 promoter region in Chinese polycystic ovary syndrome (PCOS) patients. Methods: A case-control study was designed that involved 10 PCOS patients and 10 controls. Ovary tissues obtained from 10 women with PCOS and 10 healthy controls were matched for body mass index and age. Methylation of CYP19A1 promoter was detected by methylation-specific PCR. CYP19A1 expression was measured by real-time PCR and Western blotting. Results: The methylation level of CYP19A1 promoter in PCOS samples was significantly higher than in controls (0.698 ± 0.192 vs. 0.210 ± 0.064, p < 0.01). A significant downregulation of CYP19A1 mRNA and protein expression levels was observed in PCOS ovary tissues. Furthermore, the scatter plot revealed that promoter methylation was inversely correlated with CYP19A1 mRNA level (Pearson’s correlation –0.820, p < 0.01). Conclusion: CYP19A1 expression is frequently repressed in PCOS ovaries due to the promoter hypermethylation. CYP19A1 promoter hypermethylation may play a key role in the pathogenesis of PCOS. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0378–7346/13/0764–0209$38.00/0 E-Mail [email protected] www.karger.com/goi
Introduction
Polycystic ovary syndrome (PCOS) is a common heterogeneous disorder in women of child-bearing age, and the most common cause of female infertility due to anovulation [1, 2]. Whilst the pathogenesis of PCOS remains largely unknown, accumulating data suggest that PCOS is a complex disease which involves both genetic and environmental factors [3–5]. PCOS may begin in utero, because female mammals (rhesus monkeys, sheep, mice or rats) exposed to androgen in utero develop masculinized phenotypes similar to PCOS in adulthood [6– 8]. Obesity in adolescents likely exacerbates the signs of this condition [9, 10]. Diet and exercise, such as a wellbalanced diet with restricted caloric intake and portion size, combined with regular and moderate physical activity, are recommended as the first-line treatment for PCOS by the majority of endocrinologists and gynecologists [11, 12]. Therefore, it is postulated that environmental factors may reprogram the genome, causing persistent DNA methylation modification and irregular gene expression predisposing to PCOS [13]. The cytochrome P450 family 19 subfamily A polypeptide 1 gene (CYP19A1) encodes aromatase (EC 1.14.14.1), a key steroidogenic enzyme that catalyzes the final step of Dr. Wei Zhang Obstetrics and Gynecology Hospital, Fudan University 413 Zhaozhou Road Shanghai 200011 (PR China) E-Mail weizhang 611 @ 163.com
Downloaded by: NYU Medical Center Library 128.122.253.228 - 5/14/2015 4:00:32 PM
Key Words Polycystic ovary syndrome · CYP19A1 · Methylation
Materials and Methods Patient Selection and Sample Collection Ten PCOS patients with 10 controls were recruited from the Obstetrical and Gynecological Hospital of Fudan University. All participants were of Chinese Han ethnicity. The study was approved by the institutional ethics committee and informed consent was obtained from all subjects participating in the study. PCOS patients were selected from those who underwent ovarian drilling for ovulation induction and were diagnosed based on the 2003 Rotterdam Criteria [18]. Controls were 10 normal menstruating women without signs of hyperandrogenism who underwent laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain or oophorectomy for nonovarian indications. Endocrine parameters in control subjects were all within the normal range. Ovarian tissue samples were collected from all participants, histologically examined and stored in liquid nitrogen. For each PCOS subject, a control subject was matched for age (±3 years) and body mass index (BMI; ±3) to statistically adjust for these two variables. Exclusion criteria for the study included hyperprolactinemia, thyroid disease, adrenal hyperplasia, Cushing’s syndrome, androgen-secreting tumors, endometriosis, known cardiovascular disease, neoplasms, diabetes, hypertension (blood pressure >140/90 mm Hg) and other conditions that may affect the results. None of the subjects had taken medications for at least 3 months before the study, including oral contraceptives, glucocorticoids, ovulation induction agents, antidiabetic and antiobesity drugs, or estrogenic, antiandrogenic or antihypertensive medication. Bisulfite Modification and Methylation-Specific PCR Bisulfite modification of 1 μg genomic DNA was performed by using an EZ DNA methylation kit (Zymo Research, Orange, Calif., USA) according to the manufacturer’s instructions and eluted into a 20-μl volume, which was then diluted to give a final concentration of 20 ng/μl. One microliter of bisulfite-treated genomic DNA was amplified using GC Rich DNA polymerase (Qiagen, Hilden, Germany) and the following primers: methylation-specific primers, forward 5′-GTTTAATTCGGTTAGGAAGGAGC-3′, reverse 5′-CGACTTACCCGAAACGTATAAAA-3′, 294 bp; unmethyl-
210
Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
ation primers, forward 5′-GGTTTAATTTGGTTAGGAAGGA GT-3′, reverse 5′-ACAACTTACCCAAAACATATAAAA-3′, 296 bp. PCR was carried out using an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA) under the following conditions: 1 cycle of 95 ° C for 5 min, followed by 35 cycles of denaturing at 94 ° C for 45 s, annealing at 60 ° C for 45 s, extension at 72 ° C for 45 s and final extension at 72 ° C for 7 min. PCR products were analyzed by electrophoresis on 2% agarose gel. Lymphocyte DNA purified from peripheral blood of a healthy voluntary donor was used as the negative unmethylation control. In addition, the DNA was treated in vitro with SssI-CpGMethylase (NEB, Frankfurt, Germany) and used as a positive methylation control. The optical density values for each amplified product corresponded to the subtraction of background values from medium band intensity. The extent of methylation was semiquantitatively scored by the summation of: mean optic density of the methylated PCR product/(mean optic density of the methylated PCR product + mean optic density of the unmethylated PCR product), and the final score was defined as the methylation level.
Quantitative Reverse-Transcription PCR Total RNA from individual ovarian tissues or granulose cells was isolated using Trizol (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer’s instructions. Two micrograms of total RNA was used as a template for RT-PCR. Single-stranded complementary DNA was transcribed using a PrimeScript® RTPCR Kit (Perfect Real-Time, TAKARA, Dalian, China). Quantitative RT-PCR was performed using Fast Start DNA Master SYBR Green I kit (Roche, Mannheim, Germany) with the following primers: CYP19A1, forward 5′-GCTGGAAATGATCTTTACC CCA-3′ and reverse 5′-TGTAGCCTGGTTCTCTGGTGTG-3′; GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCA CCCTGTTGCTGTA-3′. Western Blot Analysis Human ovarian tissues were homogenized as described previously [19]. The homogenates were centrifuged at 12,000 g at 4 ° C for 20 min and the protein concentration in the supernatants was estimated using the Lowry method [20], as described before [19]. Bands were detected by using enhanced chemiluminescence assay and analyzed by densitometry using the Image Pro-Plus 3.0.1 system (Media Cybernetics, Silver Spring, Md., USA) with β-actin as the loading control.
Statistical Analysis Data were expressed as mean ± SD and analyzed using SPSS software version 16.0 (SPSS, Chicago, Ill., USA). Paired comparisons were performed using the paired, two-sided Student t test. The associations between variables were accessed using Pearson’s correlation test. p < 0.05 was considered as statistically significant.
Results
The General Characteristics of PCOS Patients and Controls The general characteristics of PCOS patients and controls are shown in table 1. No significant difference beYu/Sun/Liu/Li/Wang/Zhang
Downloaded by: NYU Medical Center Library 128.122.253.228 - 5/14/2015 4:00:32 PM
estrogen biosynthesis by converting testosterone to estradiol [14]. The production of estrogen in the ovary is essential for follicular growth and selection [15]. Alterations in these processes could at least in part be responsible for the development of follicular cysts [16]. PCOS is observed in patients with aromatase deficiency due to rare loss-of-function mutations [17]. Therefore, CYP19A1 may act as a genetic modifier of the hyperandrogenic phenotype of PCOS. In this study, we speculated that the methylation of the CYP19A1 promoter region may be associated with decreased aromatase activity and hyperandrogenism in PCOS women. To test this hypothesis, we compared the methylation of CYP19A1 promoter and the expression level of CYP19A1 in PCOS patients and controls.
trol group
300 bp M
M.Sssl
U
M
Lymphocyte
U P1
1.00
0.604 0.924 0.588 0.012* 0.601 0.001* 0.870 0.024* 0.040* 0.020*
* p < 0.05.
tween age and BMI was seen between the two groups. Women with PCOS had higher levels of testosterone and luteinizing hormone (LH; p < 0.01) with higher LH/follicle-stimulating hormone (FSH) ratio (p < 0.05) and larger ovarian volume (p < 0.05) when compared to women without PCOS. The estrogen to androgen (E2/T) ratio was significantly lower in PCOS than that in control (p < 0.05). CYP19A1 Promoter Methylation Level in PCOS Patients and Controls Methylation-specific PCR (MSP) results showed that the promoters were methylated in all the investigated ovarian tissue samples (fig. 1). The relative methylation level of PCOS samples was 0.698 ± 0.192, while that of controls was 0.210 ± 0.064. A significant difference in the CYP19A1 promoter methylation level between PCOS and control groups was seen (p < 0.01). CYP19A1 mRNA Expression Level in PCOS Patients and Controls Real-time RT-PCR analysis showed that the CYP19A1 mRNA expression level was significantly lower in PCOS patients than in controls. The ratio of CYP19A1/GAPDH mRNA was 0.154 ± 0.012 in the PCOS group and 0.514 ± 0.132 in control subjects (p < 0.01; fig. 2).
M
U P2
M
U C1
M
U C2
p < 0.01
0.80
Marker
0.60 0.40 0.20 0
b
Control
PCOS
Fig. 1. MSP analysis of the methylation of CYP19A1 promoter in the two groups. a Representative gel image of MSP; MSP products: U represents the unmethylated product (296 bp); M represents the methylated product (294 bp); Lymphocyte represents the negative unmethylation control; M.SssI represents a positive methylation control; P1 and P2 represent two randomly selected samples in the PCOS group, and C1 and C2 represent two randomly selected samples in the control group. b The methylation of CYP19A1 promoter was semiquantitatively scored as the methylation level, which was 0.698 ± 0.192 for the PCOS group, significantly higher than 0.2098 ± 0.0642 in the control group (n = 10; p < 0.01).
p < 0.01
0.60
0.40
0.20
0
Control
PCOS
CYP19A1 Protein Expression Level in PCOS Patients and Controls Western blot analysis showed that the expression level of CYP19A1 protein was significantly lower in PCOS pa-
Fig. 2. Quantitative RT-PCR analysis of CYP19A1 mRNA expression in the two groups. The relative mRNA level of CYP19A1 was normalized to that of GAPDH. The ratio of CYP19A1/ GAPDH mRNA was 0.154 ± 0.012 in the PCOS group, significantly lower than 0.514 ± 0.132 in the control group (n = 10; p < 0.01).
CYP19A1 Expression Is Repressed by Promoter Hypermethylation in PCOS
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10 29.4±3.7 22.5±3.0 6.6±2.9 5.2±2.0 47.7±22.8 0.4±0.1 11.7±5.5 0.9±0.5 136.0±75.1 7.2±2.8
a
M
Color version available online
10 28.4±4.7 22.7±3.4 7.2±1.4 14.9±10.2 43.0±13.0 0.7±0.20 12.2±7.4 2.1±1.4 71.4±44.4 13.7±5.3
p value
Relative mRNA level of CYP19A1
Controls
Relative methylation level of CYP19A1 promoter
n Age, years BMI, kg/m2 FSH, mIU/ml LH, mIU/ml E2, pg/ml Testosterone, ng/ml Prolactin, ng/ml LH/FSH E2/T Ovarian volume, cm3
PCOS
U
Color version available online
Table 1. General characteristics of women with PCOS and the con-
P3
P4
C1
C2
C3
a
S 0.15
0.10
0.40
0.20
0 0
&RQWURO
PCOS
Fig. 3. Western blot analysis of CYP19A1 protein expression in the two groups. a Representative blots for the detection of CYP19A1 protein level; β-actin was used as a loading control. P1, P2, P3 and P4 represent four randomly selected samples in the PCOS group; C1, C2, C3 and C4 represent four randomly selected samples in the control group. b Densitometric analysis of CYP19A1 protein level; the relative protein level of CYP19A1 was normalized to that of β-actin. The ratio of CYP19A1/β-actin protein was 0.037 ± 0.009 in the PCOS group, significantly lower than 0.134 ± 0.031 in control group (n = 10; p < 0.01).
tients than in the controls. The ratio of CYP19A1/β-actin protein level was 0.037 ± 0.009 in the PCOS group and 0.134 ± 0.031 in the control group (p < 0.01; fig. 3). Correlation between CYP19A1 Promoter Methylation and mRNA Expression Levels The scatter plot revealed that the level of methylation was inversely correlated with CYP19A1 mRNA level, and Pearson’s correlation coefficient was –0.820 (p < 0.01; fig. 4). The results showed a significant correlation between the promoter methylation and mRNA expression level of CYP19A1.
Discussion
Methylation modification of DNA is a mechanism by which the environmental factors affect the gene expression without altering the genetic code [21]. Emerging ev212
PCOS Control
0.60
0.05
0
b
Pearson’s correlation coefficient = –0.820 p < 0.01
Gynecol Obstet Invest 2013;76:209–213 DOI: 10.1159/000355314
0.20
0.40
0.60
0.80
1.00
Relative methylation level of CYP19A1 promoter
Fig. 4. Scatter plot of CYP19A1 mRNA expression level versus pro-
moter methylation level in PCOS patients. The methylation level was inversely correlated with the CYP19A1 mRNA expression level. Pearson’s correlation coefficient was –0.820 (p < 0.01).
idence from prenatally androgenized models provides new insights into the pathogenesis of PCOS and suggests that environmental factors may predispose individuals to this disorder [4]. Moreover, the inactivation of the X chromosome and androgen receptor methylation associate with the manifestation of PCOS phenotypes [22, 23]. In this case study, we investigated the methylation of the CYP19A1 promoter in PCOS ovaries. Our results showed that the CYP19A1 promoter methylation level was significantly higher in PCOS samples than in controls. The E2/T ratio was used as an indirect measure for aromatase activity [24]. Compared with controls, PCOS had lower levels of E2/T (p < 0.05), and CYP19A1 mRNA and protein levels were both significantly lower in PCOS ovaries than in control samples. Moreover, Pearson’s correlation test showed that CYP19A1 gene promoter methylation level was inversely correlated with CYP19A1 mRNA level. Based on the present data we believe that reduced CYP19A1 expression in PCOS ovaries may be due to gene repression by DNA methylation of the CYP19A1 promoter. Ovarian androgen overproduction is a key pathologic feature of PCOS [25]. The enzyme aromatase encoded by the CYP19A1 gene mediates the conversion of the anYu/Sun/Liu/Li/Wang/Zhang
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5HODWLYHSURWHLQOHYHORICYP19A1
0.20
C4
Color version available online
P2
Relative mRNA level of CYP19A1
P1
Color version available online
CYP19A1 (58 kDa) DŽ$FWLQ (43 kDa)
drogens testosterone and androstenedione to the estrogens, E2, and estrone. Our findings suggest that methylation modification is a factor that affects aromatase expression and activity and thus influences estrogen biosynthesis. In conclusion, we demonstrated that the presence of methylation of the CYP19A1 promoter is higher in PCOS patients and correlates with the lower aromatase expression. These findings suggest that CYP19A1 expression is frequently repressed in PCOS ovaries due to
the promoter hypermethylation. CYP19A1 promoter hypermethylation may play a key role in the pathogenesis of PCOS. Acknowledgements We are extremely grateful to all the women who agreed to participate in our study. We thank Mr. Kun Guo of the Liver Cancer Institute, Zhongshan Hospital, Fudan University, for his helpful advice.
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