CYP17 Genotypes Differ in Salivary 17-B Estradiol Levels: A Study Based on Hormonal Profiles from Entire Menstrual Cycles

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1 2131 CYP17 Genotypes Differ in Salivary 17-B Estradiol Levels: A Study Based on Hormonal Profiles from Entire Menstrual Cycles Grazyna Jasienska, 1,3 Maria Kapiszewska, 2 Peter T. Ellison, 4 Malgorzata Kalemba-Drozdz, 2 Ilona Nenko, 1 Inger Thune, 5,6 and Anna Ziomkiewicz 1 1 Department of Epidemiology and Population Studies, Collegium Medicum and 2 Department of General Biochemistry, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland; 3 Radcliffe Institute for Advanced Study and 4 Department of Anthropology, Harvard University, Cambridge, Massachusetts; 5 Department of Community Medicine, University of Tromso, Norway; and 6 Ulleval University Hospital, Oslo, Norway Abstract Variation in the levels of sex-steroid hormones results from differences in developmental conditions, adult lifestyle, and genetic polymorphism. Genes involved in sex-steroid biosynthesis have been implicated to influence levels of hormones in premenopausal women, but the results were inconclusive. We tested variation among women in levels of salivary estradiol (E 2 ) corresponding to CYP17 genotypes. CYP17 encodes cytochrome P450c17A, which mediates two enzymes important in E 2 synthesis. In contrast to the earlier studies that relied on one or a few samples for assessing the E 2 levels of an individual woman, our study is based on daily collected saliva samples for one entire menstrual cycle. Sixty Polish women, ages 24 to 36 years, with regular menstrual cycles and no reported fertility problems participated in the study. Women with A2/A2 genotype had 54% higher mean E 2 levels than women with A1/A1 genotype (P = ) and 37% higher than women with A1/A2 genotype (P = ). Heterozygous A1/A2 women had 13 % higher E 2 levels than homozygous A1/A1 women (but this difference was significant only in a nonparametric test). Levels of E 2 during the day with highest E 2 (day 1) were 72% higher in A2/A2 compared with A1/A1 (P = 0.01) and 52 % higher compared with A1/A2 (P = 0.03). Our results suggest that CYP17 genotype may serve as a biomarker of endocrine function in women of reproductive age. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2131 5) Introduction Sex-steroid hormones are implicated in the development and growth of breast cancer and most of the risk factors for that disease exert their effect by influencing levels of sex-steroid hormones, especially estrogens (1-4). Levels of sex-steroids are also important for fecundity, risks of osteoporosis, cardiovascular health, and psychological well-being (5-9). Considerable variation has been documented in the levels of sex-steroid hormones among populations and among healthy, premenopausal women within a population (10). Many sources of this variation have been identified. Levels of ovarian hormones are sensitive to factors related to fetal development (11, 12), childhood growth (13), and adult life (14-22). It is also likely that variation in sex-steroid levels results partly from genetic variation (i.e., polymorphism of genes that control steroid hormone biosynthesis; refs ). The gene CYP17 encodes cytochrome P450c17a, which mediates the activity of 17a-hydroxylase and 17,20-lyase, both involved in the biosynthesis of estradiol (E 2 ; ref. 29). In women, CYP17 is expressed in the ovarian theca cells, the corpus luteum, adrenals, and adipose tissue (30-32). A single nucleotide polymorphism in the 5 -untranslated region of CYP17 is relatively common and the presence of the A2 allele is thought to increase transcription rates (26), although other Received 5/30/06; revised 8/28/06; accepted 9/13/06. Grant support: State Committee for Scientific Research, Poland and Radcliffe Institute for Advanced Study at Harvard University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Grazyna Jasienska, Department of Epidemiology and Population Studies, Collegium Medicum, Jagiellonian University, Grzegórzecka 20, Kraków, Poland. Phone: jasienska@post.harvard.edu Copyright D 2006 American Association for Cancer Research. doi: / epi studies suggest that no additional transcription factor binding activity is associated with the presence of the A2 allele (33, 34). Studies investigating the relationship between CYP17 polymorphism and levels of E 2 in premenopausal women (24, 25, 27, 35-37) have yielded inconsistent results. E 2 levels measured around day 11 of the menstrual cycle have been reported to be 11% and 57% higher among women with genotypes A1/A2 and A2/A2, respectively, compared with A1/A1 women (24). In the same study, E 2 levels during the luteal phase, around day 22 of the cycle, were reported to be 7% and 28% higher for women with A1/A2 and A2/A2, respectively. Another study found that women with the A2/A2 genotype had 42% and heterozygotes 19% higher E 2 than the A1/A1 genotype but only among women with body mass index values 25 kg/m 2 and under (25). Among women with higher body mass indexes, the genotypes did not differ in E 2 levels. Both of these studies are based on only one or two E 2 values per woman. A third study of 173 premenopausal women did not find any differences in E 2 levels among CYP17 genotypes but documented significant differences in the levels of steroid hormone dehydroepiandrosterone, a precursor for E 2 synthesis (37). E 2 levels were measured in a single blood sample presumably collected in the luteal phase of the cycle, between day 20 and 24 from the beginning of the cycle. Two other studies that did not find statistically significant differences in E 2 levels among CYP17 genotypes also used a single blood sample for hormonal measurements (35, 36). Only one study attempted to control the within-cycle variability in E 2 levels by sampling, on the average, 4.4 days per woman over a 2-year period, but did not find significant differences in relation to the CYP17 genotype (27). We document, for the first time, a relationship between polymorphism in CYP17 and full cycle profiles of 17-h E 2 among healthy, regularly menstruating women in midreproductive years. In contrast to the earlier studies that relied on

2 2132 CYP17 Polymorphism and Entire Cycle Estradiol Profiles one or a few samples for assessing the E 2 levels of an individual woman, our study is based on daily collected saliva samples for one entire menstrual cycle. Materials and Methods Study Group. The subjects were 60 urban (n = 22) and rural (n = 38) women from Poland recruited for the study by advertisements. Women were selected for participation if they met the following criteria: age between 24 and 36 years, regular menstrual cycles and no fertility problems, no gynecologic and/or chronic disorders (i.e., diabetes and hypothyroidism/ hyperthyroidism), not taking any hormonal medication or using hormonal contraception during the 6 months before recruitment, and not being pregnant or lactating during the 6 months before recruitment. The recruited women signed a consent form after being informed about the aims and requirements of the study, which had been approved by the Jagiellonian University Research Ethics Committee. Anthropometric Measurements, General Questionnaire, and Birth Characteristics. Subjects body weight, height, and percentage body fat (by bioimpedance) were measured by a trained anthropologist. A general questionnaire (one part completed by interview and one part self-administered) was used to collect information on education, reproductive history, and past use of hormonal medication, tobacco, and alcohol. Data on birth weight and birth length were recorded at birth by a nurse and obtained from subjects personal health books issued by hospitals following birth. Detailed description of the methods was published elsewhere (11, 17). E 2 Indices and Assay Procedure. Women collected daily morning saliva samples for one entire menstrual cycle. Saliva samples from 20 days (reverse cycle days 5 to 24, where the last day of each cycle was marked as day 1) of each cycle were analyzed for the concentration of E 2 using an I-125 based RIA kit (Diagnostic Systems Laboratories, Webster, TX) with published (16) modifications to the manufacturer s protocol. The sensitivity of the E 2 assay is 4 pmol/l. Average intra-assay variability was 9%, and inter-assay variability ranged from 23% for lower (15 pmol/l) to 13% for higher (50 pmol/l) values. Before other statistical analyses, cycles were aligned based on identification of the day of the midcycle E 2 drop (day 0; Fig. 1), which provides a reasonable estimate of the day of ovulation (5). E 2 values from 18 consecutive days of each cycle aligned on day 0 were used in analyses. Reliable identification of the day of the midcycle E 2 drop could not be made for two subjects. Therefore, we used data from 58 women in statistical analyses. Genotype Determination. Genotyping was done according to published methods (38). Genomic DNA was extracted from 0.2 ml peripheral blood using extraction kit (Qiagen GmbH, Hilden, Germany). PCR was done using primers that amplify restriction sites for Msp A1 I (A2 polymorphism- CYP17): 5 -CAAGGTGAAGATCAGGGTAG-3 (forward) and 5 -GCTAGGGTAAGCAGCAAGAG-3 (reverse). A 145-bp fragment encompassing biallelic single gene polymorphism (T!C) in the untranslated 5 region of CYP17 was amplified. For the PCR, 25 AL final volume of reaction mixture contained the following: 200 pg of genomic DNA, 0.45 Amol/L of each primer, 2 mmol/l MgSO 4, 200 Amol/L of each deoxynucleotide triphosphate, and 1 unit plaque-forming unit polymerase. PCR cycling conditions were 94jC for 45 seconds, 57jC for 60 seconds, and 72jC for 60 seconds for a total of 35 cycles. PCR product was digested by restriction enzyme Msp A1 I for 3 hours at 37jC, separated on 4% agarose, and visualized by ethidium bromide fluorescence under UV light. The polymorphism was identified by digestion of the PCR fragment, resulting in 70- and 75-bp DNA fragments. Genotypes Figure 1. Mean E 2 profiles for CYP17 genotypes. Mean E 2 for A2/A2 genotype is 54% higher than for A1/A1 genotype and 37% higher than for A1/A2 genotype. A1/A2 heterozygote has 13% higher E 2 than A1/A1 homozygote (but this difference is significant only in a nonparametric test). 95% Confidence intervals are omitted for clarity. identified by gel electrophoresis were assigned as homozygous wild-type (A1/A1) 145-bp band, heterozygous variant (A1/A2) 145-, 75-, and 70-bp bands, and homozygous variant (A2/A2) 75- and 70-bp bands. The protocol was run twice for each DNA sample. Additionally, 20% of templates were rerun according to an alternative method (39). A control sample which did not contain DNA was run in every reaction to confirm the absence of contamination. The quality of the PCR product was checked by running 4 ml of each sample on 1% agarose gel. Statistical Analysis. Differences among the three genotypes in mean E 2 were tested in two-way repeated measure univariate and multivariate ANOVAs. Mean values of E 2 were calculated for each woman for each 3 consecutive days of the menstrual cycle (i.e., the first arithmetic mean was calculated from untransformed values for cycle days 9, 8, and 7; the second mean from cycle days 6, 4, and 5, etc.), obtaining a six-level hormonal profile for each woman. The profile was analyzed as the repeated measure variable and the genotypes were the between-group factor (A1/A1, A1/A2, and A2/A2). The ANOVA was followed by contrast analyses; an a level of (with the Bonferroni correction) was used to indicate statistical significance. Differences among genotypes in logtransformed E 2 levels during the cycle day 1 (preovulatory day; Fig. 1) were tested in a one-way ANOVA. Differences among the genotypes in age, reproductive characteristics, length of menstrual cycle, size at birth, anthropometrics and body composition, tobacco smoking, alcohol consumption, and mean 24-hour physical activity were tested in factorial, fixed-model, one-way ANOVA analyses followed by Tukey-Kramer post-hoc tests. Results Table 1 shows the characteristics of the study group stratified by genotype. Genotypes did not show statistically significant differences in age, reproductive characteristics, size at birth, anthropometrics and body composition, tobacco smoking, alcohol consumption, and physical activity. The length of the menstrual cycle during which samples were collected did not differ among genotypes (P = 0.8). There was significant variation among genotypes in mean salivary E 2 levels (F 2,54 = 3.535; P = 0.036; see Table 2). The

3 Cancer Epidemiology, Biomarkers & Prevention 2133 Table 1. Characteristics of study subjects according to CYP17 genotype A1/A1 (N = 21), A1/A2 (N = 30), A2/A2 (N = 7), P Age (y) 28.7 (3.15) 30.2 (3.87) 29.1 (3.48) 0.33 Age at menarche (y) 13.1 (1.31) 13.1 (1.06) 13.6 (0.92) 0.53 No. children 1.9 (1.55) 1.1 (1.50) 1.1 (1.12) 0.13 Birth weight (g) 3,454.2 (528.47) 3,303.7 (697.90) 3,165.6 (507.96) 0.51 Birth length (cm) 54.3 (3.20) 53.7 (4.35) 53.4 (4.48) 0.80 Body height (cm) (6.55) (6.24) (6.66) 0.97 Body weight (kg) 62.9 (12.41) 62.8 (10.85) 59.6 (8.58) 0.74 Body mass index (kg/m 2 ) 24.0 (4.81) 24.0 (3.89) 22.7 (3.32) 0.71 Body fat (%) 28.2 (8.58) 29.4 (6.46) 27.6 (7.50) 0.78 Waist/hip ratio 0.75 (0.063) 0.74 (0.054) 0.73 (0.051) 0.46 Daily physical activity (MET-h/d) 50.1 (11.36) 48.1 (15.25) 51.0 (15.97) 0.84 Smoking (no. cigarettes/d) 2.5 (4.76) 1.6 (4.85) 4.6 (5.78) 0.31 Drinking (alcohol units/wk) 1.3 (1.0) 1.1 (0.97) 1.5 (2.16) 0.71 Self-reported usual length of a cycle (d) 28.8 (1.77) 29.1 (3.19) 28.9 (1.73) 0.94 Length of the cycle, in which saliva was sampled (d) 28.6 (3.67) 28.2 (2.81) 27.9 (3.64) 0.85 NOTE: Ps derived from factorial, fixed-model, one-way ANOVA analyses. overall interaction between the genotypes and the six-level hormonal profile was significant in the univariate repeated measure ANOVA (F 10,270 = 3.391; P = with Greenhouse- Geisser or P = with Hyun-Feldt adjustments) and marginally significant in the multivariate test [Wilks k = 0.718; degrees of freedom (df) = 10,100; P = 0.071]. Contrasts showed that the A2/A2 genotype had significantly different six-level E 2 profile from the A1/A1 genotype (F = 6.451; df = 5; P = ) and from the A1/A2 heterozygote (F = 4.347; df = 5; P = ). The heterozygote A1/A2 did not differ significantly from A1/ A1 homozygote in mean E 2 profiles (F = 0.990; df = 5; P = 0.424). However, because in the E 2 profile the heterozygote means are numerically higher than the A1/A1 homozygote means on 17 of 18 days, we did a nonparametric Wilcoxon signed-rank test to obtain an approximate statistical assessment of this difference. In this test, the profiles of A1/A1 and A1/A2 genotypes are significantly different at P = level (T = 8; N = 18), but larger sample sizes would be required to confirm this result in parametric tests. Mean E 2 on the preovulatory day 1 varied among genotypes (F 2,53 = 3.56; P = 0.03). The A2/A2 genotype had significantly higher levels of E 2 than the A1/A1 genotype (P = 0.01) and than the A1/A2 heterozygote (P = 0.03), whereas heterozygote A1/A2 did not differ significantly from the A1/A1 homozygote (P = 0.41). Discussion Our results show that variation in the levels of E 2 produced during menstrual cycles can be partially explained by polymorphism at the CYP17 locus. Women with A2/A2 genotypes had 54% higher mean E 2 levels than women with A1/A1 genotypes and 37% higher mean E 2 levels than women who had only one A2 allele. Heterozygous A1/A2 women had 13 % higher E 2 levels than homozygous A1/A1 women. Levels of E 2 during preovulatory day were 72% higher in A2/A2 compared with A1/A1 and 52% higher compared with A1/A2. Polymorphism of CYP17 gene investigated by us involves a single bp change T!C in the 5 -untranslated region at 27 bp upstream of the start of transcription. It is still controversial if this substitution influences the transcription of CYP17 or Table 2. E 2 indices according to CYP17 genotype A1/A1, A1/A2, A2/A2, Mean E 2 (pmol/l) 16.9 (8.8) 18.7 (9.69) 25.6 (12.82) Cycle day 1 (pmol/l) 29.3 (15.22) 33.2 (17.10) 50.6 (16.72) enzyme expression (33, 34, 40-44). Apparently, transcriptional efficacy is affected by other, still unknown factor(s). Our results should be treated as preliminary due to relatively small sample size, especially for the A2/A2 genotype. The frequency of the A2/A2 genotype (13%) is similar to the frequency of that genotype (14%) in the pooled sample of populations of European descent calculated from the published studies (26). Our study is the first which measured E 2 levels in daily collected saliva samples for the entire menstrual cycle. Previous studies addressed the question of variation in E 2 in menstrual cycles by measuring E 2 levels in only one or two serum or urine samples collected per woman (24, 25, 27, 35-37) and one study had, on the average, 4.4 samples per woman (27). Due to substantial intracycle variation in E 2 levels, such sampling is vastly insufficient and can lead to errors in estimating mean E 2 levels for individual women. 7 In our study, E 2 was measured in saliva reflecting free, unbound, biologically active fraction of this hormone (45). By contrast, E 2 concentrations measured in blood samples are influenced by levels of sex hormone-binding globulin. Sex hormone-binding globulin binds estrogens and regulates the bioavailability of sex steroids. Sex hormone-binding globulin has substantial interindividual variation, with additive genetic factors accounting for 68% of the total phenotypic variation (46). Urinary measurements, used in some studies, allow only for the assessment of E 2 metabolites, levels of which may be influenced by the rate of metabolic clearance, as well as genetic polymorphism in CYP1B1 or COMT involved in the 4-hydroxylation and the O-methylation of E 2 and estrone (35). Conflicting results of the previous studies may therefore result from methodologic difficulties encountered by researchers in reliable characterization of individual hormone levels. We accounted for a potential influence of lifestyle variation on E 2 levels by using selection criteria for participation and by controlling for factors known to influence levels of ovarian steroid hormones. Women in our study were in peak of reproductive years and in the age range (24-36 years) when relatively little age-related variation in levels of E 2 is expected (47). They did not use steroid contraception or other steroid medication for at least 6 months before the study. Because lactation and postpartum period are associated with changes in sex steroids, women were not recruited unless at least 6 months elapsed since these reproductive events. All women 7 G. Jasienska, M. Jasienski. Inter-population, inter-individual, inter-cycle, and intra-cycle natural variation in progesterone levels: quantitative assessment and implications for population studies, submitted for publication.

4 2134 CYP17 Polymorphism and Entire Cycle Estradiol Profiles had maintained regular menstrual cycles for at least 6 months before the study. Levels of ovarian steroid hormones produced in menstrual cycles are both important determinants of fecundity (5, 48) and are related to risk of several diseases, especially in postmenopausal years (8). Cumulative lifetime levels of sex-steroid hormones are implicated in development of hormonedependent cancers (1-4). Due to individual differences in exposure to factors modifying genetic levels of E 2, however, a relationship between CYP17 genetic polymorphism and risk of breast cancer may not be detectable. Several studies tested the relationship between CYP17 polymorphism and risk of breast cancer (23, 37, 42, 49-59), but results are conflicting. The existence of genetic variation underling variation in levels of E 2 does not preclude the importance of other factors. Well documented is the influence of lifestyle on sex-steroid levels, especially factors related to availability of metabolic energy during fetal and adult life (11, 12, 14-22, 60-62). Future studies should investigate a relationship between environmental effects and genetic polymorphism in CYP17 from the perspective of genotype-environment interactions. In addition, interactive effects of other genes involved in estrogen metabolic pathways, such as CYP19, COMT, CYP1A1, SRD5A2, and HSD17B, should be studied to account for potential epistatic interactions among genetic loci. Acknowledgments We thank the women who participated in this study, Dr. Susan F. Lipson who oversaw the steroid measurements, and students of the Faculty of Public Health, Jagiellonian University who worked as research assistants. References 1. Henderson BE, Feigelson HS. Hormonal carcinogenesis. Carcinogenesis 2000;21: Pike MC, Spicer DV, Dahmoush L, Press MF. Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. Epidemiol Rev 1993; 15: Jasienska G, Thune I, Ellison PT. Energetic factors, ovarian steroids and the risk of breast cancer. Eur J Cancer Prev 2000;9: Ellison PT. Reproductive ecology and reproductive cancers. In: Panter-Brick C, Worthman CM, editors. Hormones, Health, and Behavior A socioecological and lifespan perspective. Cambridge: Cambridge University Press; p Lipson SF, Ellison PT. Comparison of salivary steroid profiles in naturally occurring conception and non-conception cycles. Hum Reprod 1996;11: Jasienska G, Ziomkiewicz A, Gorkiewicz M, Pajak A. Body mass, depressive symptoms, and menopausal status: an examination of the jolly fat hypothesis. Womens Health Issues 2005;15: Solomon CG, Hu FB, Dunaif A, et al. Menstrual cycle irregularity and risk for future cardiovascular disease. J Clin Endocrinol Metab 2002;87: Barrett-Conor E, Bush TL. Estrogen and coronary heart disease in women. JAMA 1991;265: Riggs BL, Khosla S, Melton LJ. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 2002;23: Ellison PT, Lipson SF, O Rourke MT, et al. Population variation in ovarian function. Lancet 1993;342: Jasienska G, Ziomkiewcz A, Lipson SF, Thune I, Ellison PT. High ponderal index at birth predicts high estradiol levels in adult women. Am J Hum Biol 2006;18: Jasienska G, Lipson S, Ellison P, Thune I, Ziomkiewicz A. Symmetrical women have higher potential fertility. Evol Hum Behav 2006;27: Nunez-De-la Mora A, Chatterton RT, Choudhury O, Napolitano D, Hochman J, Bentley GR. Developmental effects on salivary progesterone levels in migrant Bangladeshi women. Am J Hum Biol 2003;15: Jasienska G, Ellison PT. Energetic factors and seasonal changes in ovarian function in women from rural Poland. Am J Hum Biol 2004;16: Jasienska G, Ellison PT. Physical work causes suppression of ovarian function in women. Proc R Soc Lond B Biol Sci 1998;265: Jasienska G, Ziomkiewicz A, Ellison PT, Lipson SF, Thune I. Large breasts and narrow waists indicate high reproductive potential in women. Proc R Soc Lond B Biol Sci 2004;271: Jasienska G, Ziomkiewicz A, Thune I, Lipson SF, Ellison PT. Habitual physical activity and estradiol levels in women of reproductive age. Eur J Cancer Prev 2006;15: Ellison PT, Lager C. Moderate recreational running is associated with lowered salivary progesterone profiles in women. Am J Obstet Gynecol 1986;154: Lager C, Ellison PT. Effect of moderate weight loss on ovarian function assessed by salivary progesterone measurements. Am J Hum Biol 1990;2: Bullen BA, Skrinar GS, Beitins IZ, von Mering G, Turnbull BA, McArthur JW. Induction of menstrual disorders by strenuous exercise in untrained women. N Engl J Med 1985;312: Pirke KM, Schweiger V, Lemmel W, Krieg JC, Berger M. The influence of dieting on the menstrual cycle of healthy young women. J Clin Endocrinol Metab 1985;60: De Souza MJ, Miller BE, Loucks AB, et al. High frequency of luteal phase deficiency and anovulation in recreational women runners: blunted elevation in follicle-stimulating hormone observed during luteal-follicular transition. J Clin Endocrinol Metab 1998;83: Haiman CA, Hankinson SE, Spiegelman D, et al. Relationship between a polymorphism in CYP17 with plasma hormone levels and breast cancer. Cancer Res 1999;59: Feigelson HS, Shames LS, Pike MC, Coetzee GA, Stanczyk FZ, Henderson BE. Cytochrome P450c17a gene (CYP17) polymorphism is associated with serum estrogen and progesterone concentrations. Cancer Res 1998;58: Small CM, Marcus M, Sherman SL, Sullivan AK, Manatunga AK, Feigelson HS. CYP17 genotype predicts serum hormone levels among pre-menopausal women. Hum Reprod 2005;20: Sharp L, Cardy AH, Cotton SC, Little J. CYP17 gene polymorphisms: prevalence and associations with hormone levels and related factors. A HuGE review. Am J Epidemiol 2004;160: Lurie G, Maskarinec G, Kaaks R, Stanczyk FZ, Le Marchand L. Association of genetic polymorphisms with serum estrogens measured multiple times during a 2-year period in premenopausal women. Cancer Epidemiol Biomarkers Prev 2005;14: Tworoger SS, Chubak J, Aiello EJ, et al. Association of CYP17, CYP19, CYP1B1, and COMT polymorphisms with serum and urinary sex hormone concentrations in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2004;13: Brentano ST, Picadoleonard J, Mellon SH, Moore CCD, Miller WL. Tissuespecific, cyclic adenosine 3,5 -monophosphate-induced, and phorbol esterrepressed transcription from the human P450c 17 promoter in mouse cells. Mol Endocrinol 1990;4: Sasano H, Okamoto M, Mason JI, et al. Immunolocalization of aromatase, 17-a-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary. J Reprod Fertil 1989;85: Hanukoglu I. Steroidogenic enzymes structure, function, and role in regulation of steroid-hormone biosynthesis. J Steroid Biochem Mol Biol 1992;43: Puche C, Jose M, Cabero A, Meseguer A. Expression and enzymatic activity of the P450c17 gene in human adipose tissue. Eur J Endocrinol 2002;146: Kristensen VN, Haraldsen EK, Anderson KB, et al. CYP17 and breast cancer risk: the polymorphism in the 5 flanking area of the gene does not influence binding to Sp-1. Cancer Res 1999;59: Lin CJ, Martens JWM, Miller WL. NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17 a-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells. Mol Endocrinol 2001;15: Garcia-Closas M, Herbstman J, Schiffman M, Glass A, Dorgan JF. Relationship between serum hormone concentrations, reproductive history, alcohol consumption, and genetic polymorphisms in pre-menopausal women. Int J Cancer 2002;102: Travis RC, Churchman M, Edwards SA, et al. No association of polymorphisms in CYP17, CYP19, and HSD17 1 with plasma estradiol concentrations in 1,090 British women. Cancer Epidemiol Biomarkers Prev 2004;13: Hong CC, Thompson HJ, Jiang C, et al. Association between the T27C polymorphism in the cytochrome P450 c17 a (CYP17) gene and risk factors for breast cancer. Breast Cancer Res Treat 2004;88: Berstein LM, Imyanitov EN, Kovalevskij AJ, et al. CYP17 and CYP19 genetic polymorphisms in endometrial cancer: association with intratumoral aromatase activity. Cancer Lett 2004;207: Lai J, Vesprini D, Chu W, Jernstrom H, Narod SA. CYP gene polymorphisms and early menarche. Mol Genet Metab 2001;74: Carey AH, Chan KL, Short F, White D, Williamson R, Franks S. Evidence for a single gene effect causing polycystic ovaries and male pattern baldness. Clin Endocrinol (Oxf) 1993;38: Kadonaga JT, Carner KR, Masiarz FR, Tijian R. Isolation of CDNA-encoding transcription factor Sp1 and functional-analysis of the DNA-binding domain. Cell 1987;51: Ambrosone CB, Moysich KB, Furberg H, et al. CYP17 genetic polymorphism, breast cancer, and breast cancer risk factors. Breast Cancer Res 2003; 5:R Miyoshi Y, Ando A, Ooka M, et al. Association of CYP17 genetic polymorphism with intra-tumoral estradiol concentrations but not with CYP17 messenger RNA levels in breast cancer tissue. Cancer Lett 2003;195: Hou LF, Xu JF, Gao YT, et al. CYP17 MspA1 polymorphism and risk of biliary tract cancers and gallstones: a population-based study in Shanghai, China. Int J Cancer 2006;118:

5 Cancer Epidemiology, Biomarkers & Prevention Riad-Fahmy D, Read GF, Walker RF, Walker SM, Griffiths K. Determination of ovarian-steroid hormone levels in saliva an overview. J Reprod Med 1987;32: Ring HJZ, Lessov CN, Reed T, et al. Heritability of plasma sex hormones and hormone binding globulin in adult male twins. J Clin Endocrinol Metab 2005;90: Lipson SF, Ellison PT. Normative study of age variation in salivary progesterone profiles. J BioSoc Sci 1992;24: Baird DD, Wilcox AJ, Weinberg CR, et al. Preimplantation hormonal differences between the conception and non-conception menstrual cycles of 32 normal women. Hum Reprod 1997;12: Wu AH, Seow A, Arakawa K, Van Den Berg D, Lee HP, Yu MC. HSD17B1 and CYP17 polymorphisms and breast cancer risk among Chinese women in Singapore. Int J Cancer 2003;104: Feigelson HS, Coetzee GA, Kolonel LN, Ross RK, Henderson BE. A polymorphism in the CYP17 gene increases the risk of breast cancer. Cancer Res 1997;57: Onland-Moret NC, van Gils CH, Roest M, Grobbee DE, Peeters PHM. Cyp17, urinary sex steroid levels and breast cancer risk in postmenopausal women. Cancer Epidemiol Biomarkers Prev 2005;14: Shin MH, Lee KM, Yang JH, et al. Genetic polymorphism of CYP17 and breast cancer risk in Korean women. Exp Mol Med 2005;37: Miyoshi Y, Iwao K, Ikeda N, Egawa C, Noguchi S. Genetic polymorphism in CYP17 and breast cancer risk in Japanese women. Eur J Cancer 2000;36: Spurdle AB, Chen X, Hopper J, et al. CYP17 promoter polymorphism and breast cancer in Australian women under forty years of age. Am J Hum Genet 2000;67: Huang CS, Chern HD, Chang KJ, Cheng CW, Hsu SM, Shen CY. Breast cancer risk associated with genotype polymorphism of the estrogenmetabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility. Cancer Res 1999;59: Mitrunen K, Jourenkova N, Kataja V, et al. Steroid metabolism gene CYP17 polymorphism and the development of breast cancer. Cancer Epidemiol Biomarkers Prev 2000;9: Bergman-Jungestrom M, Gentile M, Lundin AC, Wingren S. Association between CYP17 gene polymorphism and risk of breast cancer in young women. Int J Cancer 1999;84: Dunning AM, Healey CS, Pharoah PDP, et al. No association between a polymorphism in the steroid metabolism gene CYP17 and risk of breast cancer. Br J Cancer 1998;77: Weston A, Pan CF, Bleiweiss IJ, et al. CYP17 genotype and breast cancer risk. Cancer Epidemiol Biomarkers Prev 1998;7: Kapiszewska M, Miskiewicz M, Ellison PT, Thune I, Jasienska G. High tea consumption diminishes salivary 17h-estradiol concentration in Polish women. Br J Nutr 2006;95: Panter-Brick C, Ellison P. Seasonality of workloads and ovarian function in Nepali women. Ann N Y Acad Sci 1994;709: Ibanez L, Potau N, Enriquez G, De Zegher F. Reduced uterine and ovarian size in adolescent girls born small for gestational age. Pediatr Res 2000;47:575 7.