- Email: [email protected]
A microsatellite polymorphism (tttta)n in the promoter of the CYP11a gene in Chinese women with polycystic ovary syndrome
A microsatellite polymorphism (tttta)n in the promoter of the CYP11a gene in Chinese women with polycystic ovary syndrome A (tttta)n microsatellite polymorphism in the promoter of CYP11a gene was investigated in 201 Chinese Han women with polycystic ovary syndrome (PCOS) and 147 control women. The 6/6 genotype was defined with significant difference of CYP11a polymorphism between women with PCOS and control women, and associated with greater BMI in PCOS patients, suggesting a certain role of the six-repeat allele variant in the pathogenesis of Chinese women with PCOS. (Fertil Steril威 2006;86:223– 6. ©2006 by American Society for Reproductive Medicine.)
Polycystic ovary syndrome is the most common reproductive endocrinopathy (1), characterized by polycystic ovary, excessive androgen production, chronic anovulation, and consequently infertility (2, 3). In China, this disorder is found in 50%– 60% of outpatients in gynecologic endocrinopathy clinics (4). Excessive androgen production by the ovaries has been implicated in the pathogenesis of the hyperandrogenism that characterizes the syndrome (5). Studies using freshly isolated and long cultures of theca cells have demonstrated that androgen biosynthesis is increased in PCOS compared with normal theca cells, which seems to be an intrinsic property of the theca cell (6, 7, 8). The familial aggregation of hyperandrogenemia in PCOS kindreds further suggests that this trait has a genetic basis (9). These findings have encouraged some investigators to examine the gene involved in androgen synthesis. Gharani et al. (10) found evidence for a weak linkage to the CYP11a locus. An association study revealed significant association of a pentanucleotide repeat (tttta)n polymorphism at the promoter of the CYP11a gene, which encodes the cholesterol side-chain cleavage enzyme, and serum total T levels (11). But subsequent studies didn’t support this conclusion. Daneshmand et al. (12) concluded that the (tttta)n polymorphism does not significantly influence CYP11A mRNA expression and is unlikely to play a role in PCOS. San Millan et al. (13) did not find a strong association between the pentanucleotide repeat and hirsutism. Gaasenbeek et al. (14) found nominally significant differences in Received June 2, 2005; revised and accepted December 13, 2005. Supported by the Nature Science Foundation of China (30271353/ 30572404), the Chinese Ministry of Education, the Chinese Medical Association, and the Chinese Postdoctoral Foundation. Reprint requests: XiaoKe Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China (FAX: ⫹86-451-8213 0094; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.12.037
allele and genotype frequencies in a United Kingdom casecontrol study (an excess of the pentanucleotide four-repeat allele in cases, P⫽.005), but their findings were not substantiated in the other analyses, and no relationship was seen between variation at loci and serum T levels. On the basis of these data, CYP11a remains a potentially interesting PCOS candidate worthy of further scrutiny. In the present study, we studied the (tttta)n polymorphism in the promoter region of CYP11a gene in PCOS patients and control women from the Chinese Han population to evaluate the association and then evaluated the relationship between the individual allelic variation and hyperandrogenemia or other hormone levels. The study was approved by the joint institutional review board at the Medical School of Nanjing University and Jinling Hospital, and informed consent was obtained from all participants. The diagnosis of PCOS was based on the 2003 Rotterdam European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine–sponsored symposium on PCOS (15). The criteria used included clinical and/or biochemical signs of hyperandrogenism, chronic anovulation, and polycystic ovaries, as well as the exclusion of other etiologies, such as congenital adrenal hyperplasia, androgen-secreting tumors, Cushing’s syndrome, by appropriate tests, such as serum 17-hydroxyprogesterone, cortisol determination, ultrosonography, etcl. Two hundred one patients (age 26.1 ⫾ 4.0 years) with PCOS were enrolled consecutively at the Department of Obstetrics and Gynecology of Jingling Hospital, Medical School of Nanjing University and at Anhui Medical University. On hundred forty-seven healthy women (age 29.1 ⫾ 5.1 years) who had normal menstrual cycles (⬍32 days) (most of them with one or more child) without obesity, hirsutism, acne, or overmuch sebum were enrolled and evaluated by gynecologists from the Medical School of Nanjing University. None of the patients whose hormone levels were measured had been taking hormonal medica-
Fertility and Sterility姞 Vol. 86, No. 1, July 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
223
tions, including contraceptive pills, for the previous three months. We recorded the menarche age, calculated the body mass index (BMI; kg/m2) to assess obesity. The patients’ peripheral blood was obtained by a single venipuncture during menstrual period of cycle between 8 a.m. and 9 a.m. after a 12-hour overnight fast. Samples were immediately centrifuged; serum was separated and frozen at ⫺80°C until assayed. Total T, FSH, LH, P, E2, and PRL levels in the sera of 103 PCOS patients were determined by radioimmunoassay. We failed to assay the hormone levels in all control women and the remaining 98 patients with PCOS because of the obvious economic burdens, but all had free ultrasound examinations at the department facility. Total DNA was extracted from leukocyte samples of all individuals using Chelex-100 (Promega, Madison, WI) as a medium. The DNA from all patients and controls was typed for the CYP11a (tttta)n microsatellite marker by electrophoresis of locus-specific prime polymerase chain reaction products in denaturing polyacryamide gels and subsequent detection by silver staining. The primers were designed from the published sequence of the human CYP11a gene: forward, GGT GAA ACT GTG CCA TTG C; reverse, GTT TGG GGG AAA TGA GGG GC (11). Fisher exact test was used to compare the CYP11a (tttta)n genotype distribution in the case-control study. The analysis was performed using the SAS system software (SAS Institute, Cary, NC). The results of serum hormone levels are reported as mean ⫾ SD. Differences in serum hormone levels were assessed using one-way analysis of variance. P⬍.05 was considered statistically significant. Hardy-Weinberg distribution of genotypes in the PCOS and control groups was assessed.
FIGURE 1 Electrophoresis on a 6% denaturing polyacrylamide gel of polymerase chain reaction– amplified fragments from the gene CYP11a 5= regulatory region, showing the various alleles of the microsatellite polymorphism (ttta)n. Lane 1: bands of (ttta)4 and (ttta)9, indicating genotype 4/9. Lane 2: bands of (ttta)6 and (ttta)9, indicating genotype 6/9. Lane 3: bands of (ttta)6 and (ttta)7, indicating genotype 6/7. Lane 4: bands of (ttta)8 and (ttta)9, indicating genotype 8/9. Lane 5: bands of (ttta)8 and (ttta)10, indcating genotype 8/10. Lane 6: bands of (ttta)6 and (ttta)10, indicating genotype 6/10. The sizes of the fragments were estimated using the allele ladder (lane AL). The gel was silver stained.
Wang. CYP11a polymorphism and PCOS. Fertil Steril 2006.
PCOS groups in basal FSH, LH, LH/FSH, total T, P, E2, and PRL or in menarche age.
Seven different alleles were identified, corresponding to 4-, 6-, 7-, 8-, 9-, 10-, and 11-repeat-units alleles (Fig. 1). The genotype distribution of polymorphism in both groups was in Hardy-Weinberg equilibrium in the case-control study (P⫽.298 and P⫽.749, respectively). After multiple comparisons, the results show differences of individual genotype distribution in the case-control study (Fisher exact test [two-tailed], P⫽.031) (Table 1). The percentages of 4/8 and 6/6 genotypes were statistically different between women with PCOS and control women (4/8: 1.5% and 6.1%, respectively, 2⫽5.467, P⫽.019; 6/6: 57.7% and 32.8%, respectively, 2⫽5.588, P⫽.018) (Table 1). No differences were observed in the distribution of other genotypic individuals (Table 1).
When all the subjects with PCOS were divided into those having at least one copy of the common four-repeat-units allele (4R⫹ genotype) and those with no four-repeat-units allele (4R⫺ genotype) (10, 11), no statistical differences were observed in the genotype distribution (2 ⫽ 2.933, P⫽.087), BMI, or hormone levels. However, When all PCOS subjects were divided into those having at least one copy of the common six-repeat-units allele (6R⫹ genotype) and those with no six-repeat-units allele (6R⫺ genotype), the BMI of 6R⫹ genotype individuals was significantly higher than that of 6R⫺ genotype individuals (P⫽.0075); no differences were observed in the genotype distribution and hormone levels.
The 4/4 genotype patients had lower BMI (19.4 ⫾ 1.7 kg/m2) than those with variants containing six-repeat-units alleles, including the 4/6, 6/6, and 6/8 genotypes (23.6 ⫾ 3.3, 22.5 ⫾ 3.8, and 22.2 ⫾ 4.2 kg/m2, respectively) (P⫽.0037, 0.0027, and 0.059, respectively). No statistical differences were observed among the different genotypic
Our results indicated that there is an association between the general CYP11a polymorphisms and PCOS. Specifically, the percentage of 4/8 genotype was statistically lower in women with PCOS than in controls, suggesting that genotype 4/8 may protect women against PCOS. However, the total number of 4/8 patients investigated is too small,
224
Wang et al.
Correspondence
Vol. 86, No. 1, July 2006
As yet no insight was provided to explain how the 6/6 genotype might affect CYP11a function and thus PCOS expression. Further investigation of the promoter region of CYP11a is required to determine the molecular basis of association between the (tttta) repeat polymorphism and the expression of this gene. Our present study indicated an effect of this allelic variant on higher BMI of women with PCOS. Because Cyp11a is closely associated with the production of androgen, we suppose that such an effect in PCOS is achieved by interference of androgens to carbohydrate metabolism and insulin resistance. Comparing with the previous study, we discovered that the allele distribution of CYP11a is quite different among various populations. We identified seven allele fragments in Chinese Han women, whereas previous studies found only four allele fragments in women of different populations. In Chinese Han woman (tttta)6 (6R) had the highest frequency (71%), whereas in Spanish, Greek, British, or other European women (tttta)4 (4R) is the most common allele (54%, 53%, 58.2%, 59%, respectively). Thus ethnicity could explain this common genotypic difference, whereas a big sample size is related to some rare allelic discoveries.
Wang. CYP11a polymorphism and PCOS. Fertil Steril 2006.
Note: The genotype distribution of both polymorphisms was in Hardy-Weinberg equilibrium in the case-control study (P⫽.298 and P⫽.749, respectively). a Fisher exact test (2-tailed), P⫽.031, PCOS vs. control.
0 1 .242 0 1 .242 2 0 .225 0 1 .242 1 0 .392 3 9 .019 Study group PCOS (n ⫽ 201) Control (n ⫽ 147) P valuesa
11 5 .360
44 41 .162
0 1 .242
116 66 .023
1 1 .824
24 20 .644
0 1 .242
8/10 8/9 8/8 6/10 6/9 6/8 6/7 6/6 4/11 4/9 4/8 4/6 4/4
Genotype
CYP11a (tttta)n microsatellite polymorphism genotype analysis of patients with PCOS and healthy women.
TABLE 1
Fertility and Sterility姞
and whether the 4/8 genotype really has a protective role needs further study. As for the 6/6 genotype, its frequency in PCOS individuals was statistically higher than that of control subjects, although it is the most common variant of CYP11a (tttta)n microsatellite polymorphism. Individual numbers of 6/6 was enough that we could define the 6/6 genotype in CYP11a polymorphisms as indicating increased risk for Chinese women to develop PCOS.
As we know, there are some clinical differences between PCOS women with various ethnic origins. Menstrual abnormalities and infertility are more severe in patients of South Asian origin than of European origin, whereas obesity and androgenic signs are more common in patients of the latter origin (16). As we have shown here, the ethnic difference of CYP11a polymorphism could make some contribution in this regard. In conclusion, 6R allele is the most common fragment in the Chinese population. The 6/6 genotype has been defined as significantly different CYP11a microsatellite polymorphism between PCOS and control subjects, and the sixrepeat-units allele genotypes are associated with greater BMI in PCOS patients, indicating an involvement of this allelic variant, CYP11a (tttta)6 marker, in the pathogenesis of Chinese women with PCOS. Yong Wang, Ph.D.a,b XiaoKe Wu, M.D., Ph.D.b Yunxia Cao, M.D., Ph.D.c Long Yi, Ph.D.a Jianxiu Chen, B.Sc.d 225
a
Human Genetic Identification Laboratory, Medical School of Nanjing University, Nanjing, China; b Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, China; c Department of Obstetrics and Gynecology, Anhui Medical University, Hefei, China; and d School of Life Science, Nanjing University, Nanjing, China
9.
10.
11.
REFERENCES 1. Wu XK, Zhou SY, Liu JX, Pollanen P, Sallinen K, Makinen M, et al. Selective ovary resistance to insulin signaling in women with polycystic ovary syndrome. Fertil Steril 2003;80:954 – 65. 2. Dunaif A, Givens JR, Haseltine F, Merriam GR, editors. The polycystic ovary syndrome. Cambridge (MA): Blackwell Scientific; 1992. 3. Wood JR, Nelson VL, Ho C, Jansen E, Wang CY, Urbanek M, et al. The molecular phenotype of polycystic ovary syndrome (PCOS) theca cells and new candidate PCOS genes defined by microarray analysis. J Biol Chem 2003;278:26380 –90. 4. Su Y. Polycystic ovary syndrome. In: Zhang L, Clinical reproductive endocrinology. Beijing: Chinese Scientific; 2001;367–99. 5. Franks S. The ubiquitous polycystic ovary. J Endocrinol 1991;129: 317–9. 6. Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995;16:322–53. 7. Gilling-Smith C, Willis DS, Beard RW, Franks S. Hypersecretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab 1994;79:1158 – 65. 8. Nelson VL, Legro RS, Strauss JF III, McAllister JM. Augmented androgen production is a stable steroidogenic phenotype of propa-
226
Wang et al.
Correspondence
12.
13.
14.
15.
16.
gated theca cells from polycystic ovaries. Mol. Endocrinol 1999; 13:946 –57. Legro RS, Driscoll D, Strauss JF 3rd, Fox J, Dunaif A. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Natl Acad Sci USA 1998;95:14956 – 60. Gharani N, Waterworth DM, Batty S, White D, Gilling-Smith C, Conway GS, et al. Association of the steroid synthesis gene CYP11a with polycystic ovary syndrome and hyperandrogenism. Hum Mol Genet 1997;6:397– 402. Diamanti-Kandarakis E, Bartzis MI, Bergiele AT, Tsianateli TC, Kouli CR. Microsatellite polymorphism (tttta)(n) at ⫺528 base pairs of gene CYP11alpha influences hyperandrogenemia in patients with polycystic ovary syndrome. Fertil Steril 2000;73:735– 41. Daneshmand SD, Weitsman SR, Navab A. Jakimiuk AJ, Magoffin DA. Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17␣-hydroxylase/C17-20 lyase promoters. Fertil Steril 2002;77:274 – 80. San Millan JL, Sancho J, Calvo RM, Escobar-Morreale HF. Role of the pentanucleotide (tttta)(n) polymorphism in the promoter of the CYP11a gene in the pathogenesis of hirsutism. Fertil Steril 2001;75: 797– 802. Gaasenbeek M, Powell BL, Sovio U, Haddad L, Gharani N, Bennett A, et al. Large-Scale analysis of the relationship between CYP11A promoter variation, polycystic ovarian syndrome, and serum testosterone. J Clin Endocrinol Metab 89:2408 –13. The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19 –25. Williamson K, Gunn AJ, Johnson N, Milsom SR. The impact of ethnicity on the presentation of polycystic ovarian syndrome. Aust NZ J Obstet Gynaecol 2001;41:202– 6.
Vol. 86, No. 1, July 2006
Polycystic ovary syndrome is the most common reproductive endocrinopathy (1), characterized by polycystic ovary, excessive androgen production, chronic anovulation, and consequently infertility (2, 3). In China, this disorder is found in 50%– 60% of outpatients in gynecologic endocrinopathy clinics (4). Excessive androgen production by the ovaries has been implicated in the pathogenesis of the hyperandrogenism that characterizes the syndrome (5). Studies using freshly isolated and long cultures of theca cells have demonstrated that androgen biosynthesis is increased in PCOS compared with normal theca cells, which seems to be an intrinsic property of the theca cell (6, 7, 8). The familial aggregation of hyperandrogenemia in PCOS kindreds further suggests that this trait has a genetic basis (9). These findings have encouraged some investigators to examine the gene involved in androgen synthesis. Gharani et al. (10) found evidence for a weak linkage to the CYP11a locus. An association study revealed significant association of a pentanucleotide repeat (tttta)n polymorphism at the promoter of the CYP11a gene, which encodes the cholesterol side-chain cleavage enzyme, and serum total T levels (11). But subsequent studies didn’t support this conclusion. Daneshmand et al. (12) concluded that the (tttta)n polymorphism does not significantly influence CYP11A mRNA expression and is unlikely to play a role in PCOS. San Millan et al. (13) did not find a strong association between the pentanucleotide repeat and hirsutism. Gaasenbeek et al. (14) found nominally significant differences in Received June 2, 2005; revised and accepted December 13, 2005. Supported by the Nature Science Foundation of China (30271353/ 30572404), the Chinese Ministry of Education, the Chinese Medical Association, and the Chinese Postdoctoral Foundation. Reprint requests: XiaoKe Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China (FAX: ⫹86-451-8213 0094; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.12.037
allele and genotype frequencies in a United Kingdom casecontrol study (an excess of the pentanucleotide four-repeat allele in cases, P⫽.005), but their findings were not substantiated in the other analyses, and no relationship was seen between variation at loci and serum T levels. On the basis of these data, CYP11a remains a potentially interesting PCOS candidate worthy of further scrutiny. In the present study, we studied the (tttta)n polymorphism in the promoter region of CYP11a gene in PCOS patients and control women from the Chinese Han population to evaluate the association and then evaluated the relationship between the individual allelic variation and hyperandrogenemia or other hormone levels. The study was approved by the joint institutional review board at the Medical School of Nanjing University and Jinling Hospital, and informed consent was obtained from all participants. The diagnosis of PCOS was based on the 2003 Rotterdam European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine–sponsored symposium on PCOS (15). The criteria used included clinical and/or biochemical signs of hyperandrogenism, chronic anovulation, and polycystic ovaries, as well as the exclusion of other etiologies, such as congenital adrenal hyperplasia, androgen-secreting tumors, Cushing’s syndrome, by appropriate tests, such as serum 17-hydroxyprogesterone, cortisol determination, ultrosonography, etcl. Two hundred one patients (age 26.1 ⫾ 4.0 years) with PCOS were enrolled consecutively at the Department of Obstetrics and Gynecology of Jingling Hospital, Medical School of Nanjing University and at Anhui Medical University. On hundred forty-seven healthy women (age 29.1 ⫾ 5.1 years) who had normal menstrual cycles (⬍32 days) (most of them with one or more child) without obesity, hirsutism, acne, or overmuch sebum were enrolled and evaluated by gynecologists from the Medical School of Nanjing University. None of the patients whose hormone levels were measured had been taking hormonal medica-
Fertility and Sterility姞 Vol. 86, No. 1, July 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
223
tions, including contraceptive pills, for the previous three months. We recorded the menarche age, calculated the body mass index (BMI; kg/m2) to assess obesity. The patients’ peripheral blood was obtained by a single venipuncture during menstrual period of cycle between 8 a.m. and 9 a.m. after a 12-hour overnight fast. Samples were immediately centrifuged; serum was separated and frozen at ⫺80°C until assayed. Total T, FSH, LH, P, E2, and PRL levels in the sera of 103 PCOS patients were determined by radioimmunoassay. We failed to assay the hormone levels in all control women and the remaining 98 patients with PCOS because of the obvious economic burdens, but all had free ultrasound examinations at the department facility. Total DNA was extracted from leukocyte samples of all individuals using Chelex-100 (Promega, Madison, WI) as a medium. The DNA from all patients and controls was typed for the CYP11a (tttta)n microsatellite marker by electrophoresis of locus-specific prime polymerase chain reaction products in denaturing polyacryamide gels and subsequent detection by silver staining. The primers were designed from the published sequence of the human CYP11a gene: forward, GGT GAA ACT GTG CCA TTG C; reverse, GTT TGG GGG AAA TGA GGG GC (11). Fisher exact test was used to compare the CYP11a (tttta)n genotype distribution in the case-control study. The analysis was performed using the SAS system software (SAS Institute, Cary, NC). The results of serum hormone levels are reported as mean ⫾ SD. Differences in serum hormone levels were assessed using one-way analysis of variance. P⬍.05 was considered statistically significant. Hardy-Weinberg distribution of genotypes in the PCOS and control groups was assessed.
FIGURE 1 Electrophoresis on a 6% denaturing polyacrylamide gel of polymerase chain reaction– amplified fragments from the gene CYP11a 5= regulatory region, showing the various alleles of the microsatellite polymorphism (ttta)n. Lane 1: bands of (ttta)4 and (ttta)9, indicating genotype 4/9. Lane 2: bands of (ttta)6 and (ttta)9, indicating genotype 6/9. Lane 3: bands of (ttta)6 and (ttta)7, indicating genotype 6/7. Lane 4: bands of (ttta)8 and (ttta)9, indicating genotype 8/9. Lane 5: bands of (ttta)8 and (ttta)10, indcating genotype 8/10. Lane 6: bands of (ttta)6 and (ttta)10, indicating genotype 6/10. The sizes of the fragments were estimated using the allele ladder (lane AL). The gel was silver stained.
Wang. CYP11a polymorphism and PCOS. Fertil Steril 2006.
PCOS groups in basal FSH, LH, LH/FSH, total T, P, E2, and PRL or in menarche age.
Seven different alleles were identified, corresponding to 4-, 6-, 7-, 8-, 9-, 10-, and 11-repeat-units alleles (Fig. 1). The genotype distribution of polymorphism in both groups was in Hardy-Weinberg equilibrium in the case-control study (P⫽.298 and P⫽.749, respectively). After multiple comparisons, the results show differences of individual genotype distribution in the case-control study (Fisher exact test [two-tailed], P⫽.031) (Table 1). The percentages of 4/8 and 6/6 genotypes were statistically different between women with PCOS and control women (4/8: 1.5% and 6.1%, respectively, 2⫽5.467, P⫽.019; 6/6: 57.7% and 32.8%, respectively, 2⫽5.588, P⫽.018) (Table 1). No differences were observed in the distribution of other genotypic individuals (Table 1).
When all the subjects with PCOS were divided into those having at least one copy of the common four-repeat-units allele (4R⫹ genotype) and those with no four-repeat-units allele (4R⫺ genotype) (10, 11), no statistical differences were observed in the genotype distribution (2 ⫽ 2.933, P⫽.087), BMI, or hormone levels. However, When all PCOS subjects were divided into those having at least one copy of the common six-repeat-units allele (6R⫹ genotype) and those with no six-repeat-units allele (6R⫺ genotype), the BMI of 6R⫹ genotype individuals was significantly higher than that of 6R⫺ genotype individuals (P⫽.0075); no differences were observed in the genotype distribution and hormone levels.
The 4/4 genotype patients had lower BMI (19.4 ⫾ 1.7 kg/m2) than those with variants containing six-repeat-units alleles, including the 4/6, 6/6, and 6/8 genotypes (23.6 ⫾ 3.3, 22.5 ⫾ 3.8, and 22.2 ⫾ 4.2 kg/m2, respectively) (P⫽.0037, 0.0027, and 0.059, respectively). No statistical differences were observed among the different genotypic
Our results indicated that there is an association between the general CYP11a polymorphisms and PCOS. Specifically, the percentage of 4/8 genotype was statistically lower in women with PCOS than in controls, suggesting that genotype 4/8 may protect women against PCOS. However, the total number of 4/8 patients investigated is too small,
224
Wang et al.
Correspondence
Vol. 86, No. 1, July 2006
As yet no insight was provided to explain how the 6/6 genotype might affect CYP11a function and thus PCOS expression. Further investigation of the promoter region of CYP11a is required to determine the molecular basis of association between the (tttta) repeat polymorphism and the expression of this gene. Our present study indicated an effect of this allelic variant on higher BMI of women with PCOS. Because Cyp11a is closely associated with the production of androgen, we suppose that such an effect in PCOS is achieved by interference of androgens to carbohydrate metabolism and insulin resistance. Comparing with the previous study, we discovered that the allele distribution of CYP11a is quite different among various populations. We identified seven allele fragments in Chinese Han women, whereas previous studies found only four allele fragments in women of different populations. In Chinese Han woman (tttta)6 (6R) had the highest frequency (71%), whereas in Spanish, Greek, British, or other European women (tttta)4 (4R) is the most common allele (54%, 53%, 58.2%, 59%, respectively). Thus ethnicity could explain this common genotypic difference, whereas a big sample size is related to some rare allelic discoveries.
Wang. CYP11a polymorphism and PCOS. Fertil Steril 2006.
Note: The genotype distribution of both polymorphisms was in Hardy-Weinberg equilibrium in the case-control study (P⫽.298 and P⫽.749, respectively). a Fisher exact test (2-tailed), P⫽.031, PCOS vs. control.
0 1 .242 0 1 .242 2 0 .225 0 1 .242 1 0 .392 3 9 .019 Study group PCOS (n ⫽ 201) Control (n ⫽ 147) P valuesa
11 5 .360
44 41 .162
0 1 .242
116 66 .023
1 1 .824
24 20 .644
0 1 .242
8/10 8/9 8/8 6/10 6/9 6/8 6/7 6/6 4/11 4/9 4/8 4/6 4/4
Genotype
CYP11a (tttta)n microsatellite polymorphism genotype analysis of patients with PCOS and healthy women.
TABLE 1
Fertility and Sterility姞
and whether the 4/8 genotype really has a protective role needs further study. As for the 6/6 genotype, its frequency in PCOS individuals was statistically higher than that of control subjects, although it is the most common variant of CYP11a (tttta)n microsatellite polymorphism. Individual numbers of 6/6 was enough that we could define the 6/6 genotype in CYP11a polymorphisms as indicating increased risk for Chinese women to develop PCOS.
As we know, there are some clinical differences between PCOS women with various ethnic origins. Menstrual abnormalities and infertility are more severe in patients of South Asian origin than of European origin, whereas obesity and androgenic signs are more common in patients of the latter origin (16). As we have shown here, the ethnic difference of CYP11a polymorphism could make some contribution in this regard. In conclusion, 6R allele is the most common fragment in the Chinese population. The 6/6 genotype has been defined as significantly different CYP11a microsatellite polymorphism between PCOS and control subjects, and the sixrepeat-units allele genotypes are associated with greater BMI in PCOS patients, indicating an involvement of this allelic variant, CYP11a (tttta)6 marker, in the pathogenesis of Chinese women with PCOS. Yong Wang, Ph.D.a,b XiaoKe Wu, M.D., Ph.D.b Yunxia Cao, M.D., Ph.D.c Long Yi, Ph.D.a Jianxiu Chen, B.Sc.d 225
a
Human Genetic Identification Laboratory, Medical School of Nanjing University, Nanjing, China; b Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, China; c Department of Obstetrics and Gynecology, Anhui Medical University, Hefei, China; and d School of Life Science, Nanjing University, Nanjing, China
9.
10.
11.
REFERENCES 1. Wu XK, Zhou SY, Liu JX, Pollanen P, Sallinen K, Makinen M, et al. Selective ovary resistance to insulin signaling in women with polycystic ovary syndrome. Fertil Steril 2003;80:954 – 65. 2. Dunaif A, Givens JR, Haseltine F, Merriam GR, editors. The polycystic ovary syndrome. Cambridge (MA): Blackwell Scientific; 1992. 3. Wood JR, Nelson VL, Ho C, Jansen E, Wang CY, Urbanek M, et al. The molecular phenotype of polycystic ovary syndrome (PCOS) theca cells and new candidate PCOS genes defined by microarray analysis. J Biol Chem 2003;278:26380 –90. 4. Su Y. Polycystic ovary syndrome. In: Zhang L, Clinical reproductive endocrinology. Beijing: Chinese Scientific; 2001;367–99. 5. Franks S. The ubiquitous polycystic ovary. J Endocrinol 1991;129: 317–9. 6. Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995;16:322–53. 7. Gilling-Smith C, Willis DS, Beard RW, Franks S. Hypersecretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab 1994;79:1158 – 65. 8. Nelson VL, Legro RS, Strauss JF III, McAllister JM. Augmented androgen production is a stable steroidogenic phenotype of propa-
226
Wang et al.
Correspondence
12.
13.
14.
15.
16.
gated theca cells from polycystic ovaries. Mol. Endocrinol 1999; 13:946 –57. Legro RS, Driscoll D, Strauss JF 3rd, Fox J, Dunaif A. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Natl Acad Sci USA 1998;95:14956 – 60. Gharani N, Waterworth DM, Batty S, White D, Gilling-Smith C, Conway GS, et al. Association of the steroid synthesis gene CYP11a with polycystic ovary syndrome and hyperandrogenism. Hum Mol Genet 1997;6:397– 402. Diamanti-Kandarakis E, Bartzis MI, Bergiele AT, Tsianateli TC, Kouli CR. Microsatellite polymorphism (tttta)(n) at ⫺528 base pairs of gene CYP11alpha influences hyperandrogenemia in patients with polycystic ovary syndrome. Fertil Steril 2000;73:735– 41. Daneshmand SD, Weitsman SR, Navab A. Jakimiuk AJ, Magoffin DA. Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17␣-hydroxylase/C17-20 lyase promoters. Fertil Steril 2002;77:274 – 80. San Millan JL, Sancho J, Calvo RM, Escobar-Morreale HF. Role of the pentanucleotide (tttta)(n) polymorphism in the promoter of the CYP11a gene in the pathogenesis of hirsutism. Fertil Steril 2001;75: 797– 802. Gaasenbeek M, Powell BL, Sovio U, Haddad L, Gharani N, Bennett A, et al. Large-Scale analysis of the relationship between CYP11A promoter variation, polycystic ovarian syndrome, and serum testosterone. J Clin Endocrinol Metab 89:2408 –13. The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19 –25. Williamson K, Gunn AJ, Johnson N, Milsom SR. The impact of ethnicity on the presentation of polycystic ovarian syndrome. Aust NZ J Obstet Gynaecol 2001;41:202– 6.
Vol. 86, No. 1, July 2006