- Email: [email protected]
Follicle-stimulating hormone receptor and DAZL gene polymorphisms do not affect the age of menopause
MENOPAUSE Follicle-stimulating hormone receptor and DAZL gene polymorphisms do not affect the age of menopause Irene Zerbetto, M.D.,a J€ org Gromoll, Ph.D.,b Stefano Luisi, M.D., Ph.D.,a Fernando M. Reis, M.D., a Ph.D., Eberhard Nieschlag, M.D.,b Manuela Simoni, M.D., Ph.D.,b and Felice Petraglia, M.D.a a
Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy; and b Institute of Reproductive Medicine, University Hospital, M€unster, Germany
Objective: To evaluate whether single nucleotide FSH receptor and DAZL gene polymorphisms are associated with menarche and menopause timing. Design: Prospective study. Setting: Siena and M€unster Universities. Patient(s): Physiologically menopausal women. Intervention(s): The presence of FSH receptor or DAZL gene polymorphisms was investigated. Blood samples were collected and polymorphisms evaluated in extracted genomic DNA. Main Outcome Measure(s): Menarche age, menopausal age, and total years of fertility were evaluated on the basis of FSH receptor (genotypes Asn/Asn, Asn/Ser, and Ser/Ser at codon 680) and DAZL gene. Result(s): The median age of menarche was 13 years in the Asn/Asn group and 12 years in the Asn/Ser and Ser/Ser groups. The median age at menopause was 50 years in the Asn/Asn and Asn/Ser groups and 51 years in the Ser/Ser group. The length of the fertile period was 37 years in the Asn/Asn group, 38 years in the Asn/Ser group, and 39 years in the Ser/Ser group. Regarding the DAZL polymorphism, A/A, A/G, and G/G had the same age at menarche, age at menopause, and length of fertile period. Conclusion(s): We found that genotype of FSH receptor or SNP of DAZL do not predict the age at natural menopause and duration of fertility in women. The presence of Asn680/Asn680 genotype is associated with a slightly delayed age at menarche. (Fertil Steril 2008;90:2264–8. 2008 by American Society for Reproductive Medicine.) Key Words: FSH receptor, DAZL, menarche, menopause, SNP
Genetic influences have been shown to contribute greatly to the timing of puberty (1, 2) and of menopause. Menarche and menopause depend on the interaction of the hypothalamic–pituitary–ovarian axis with the female reproductive system and other endocrine organs (3). Menarche and menopause are characterized, respectively, by a physiological increase and decrease of ovarian hormone production, causing typical somatic and/or psychological alterations. In the last decade it has been shown that single nucleotide polymorphisms (SNPs) are responsible for the interindividual variation of numerous physiological functions, among them blood pressure, drug metabolism, blood clotting, and cardiovascular dysfunction (4). Preliminary studies Received June 29, 2007; revised October 18, 2007; accepted October 25, 2007. Supported by grants from the Italian Ministry of University and Scientific Research and the University of Siena. The funding source had no role in the study design, data collection, data interpretation, data analysis, or writing of the report. Reprint requests: Felice Petraglia, M.D., Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena Policlinico ‘‘Le Scotte’’ Viale Bracci, 53100 Siena, Italy (FAX: 39-0577-233-454; E-mail: [email protected]).
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have shown a correlation between SNPs in genes involved in estrogen metabolism and the timing of menarche (5), and between SNP in the estrogen receptor gene and the timing of menopause (5, 6). Follicle-stimulating hormone is a key hormone in the modulation of the hypothalamic–pituitary–ovarian axis, and its receptors are expressed in women mainly in ovarian granulosa cells (7). The FSH receptor (FSHR) belongs to the family of G protein-coupled receptors inducing signal transduction by the protein kinase A/cyclic adenosine monophosphate pathway (8). Apart from rare mutations, two common SNPs were identified in exon 10 of the FSHR gene at nucleotide positions 919 and 2039, respectively. The first SNP is located in the extracellular domain at codon position 307 (National Center for Biotechnology Information [NCBI] reference SNP ID: rs6165), which can be occupied either by alanine (Ala) or by threonine (Thr). The second SNP is located in the intracellular domain at codon position 680 and has either asparagine (Asn) or serine (Ser) (NCBI reference SNP ID: rs6166). The two SNPs are mostly in linkage disequilibrium in the Caucasian population, resulting in two discrete allelic variants, Thr307-Asn680 and Ala307-Ser680 (9), the latter
Fertility and Sterility Vol. 90, No. 6, December 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.036
representing approximately 40% of FSH receptor alleles worldwide.
were surgical menopause, known gynecological diseases, and premature ovarian failure.
The FSHR 680 polymorphism has been investigated in women undergoing controlled ovarian hyperstimulation (10, 11), in whom FSHR isoforms are involved in determining the biological effects of the hormone and may help to define the optimal FSH starting dose. Recently, different kinetics of hormonal secretion and longer duration of menstrual cycles in women with the Ser680/Ser680 genotype have been shown (12), raising the question of whether this FSHR polymorphism has any role in determining the duration of fertility and the age at which menarche and menopause occur.
The remaining subjects (n ¼ 130) were women undergoing ambulatory diagnostic procedures at the clinical chemistry laboratory of the University of Siena who volunteered to donate a blood sample for the study. These women answered a questionnaire asking for age at menarche and physiological menopause. Personal identifiers were removed from all samples. Menarche was defined as age at first menstrual period (as recalled by the study participants); natural menopause was defined as age of the last period without having further menstrual bleeding for at least 12 consecutive months.
In this field, a family of RNA binding protein, the DAZ (deleted in azoospermia) gene family, has been identified. It consists of two autosomal genes, namely BOULE and DAZL (DAZ-like), and the Y-chromosomal DAZ gene cluster. These genes are expressed exclusively in prenatal and/or postnatal germ cells and are assumed to be master regulators of germline expression (13–15). Studies in DAZL knockout mice (16) showed the complete absence of oocytes, suggesting that DAZL is involved in oocyte maturation and, possibly, in follicular exhaustion. Some DAZL polymorphisms have being associated with premature ovarian failure, although not with the age of physiological menopause (17). Of particular interest is an SNP located at the DAZL nucleotide position 260 (exon 2; A/G change), just upstream of the RNA binding domain, which leads to a threonine-to-alanine change at amino acid position 12 with possible impact on germ cell reserve (18). Whether this particular SNP is related to the duration of the fertile period in women is still unknown.
DNA Isolation and Analysis Genomic DNA was obtained from peripheral blood leukocytes using the Flexi Gene DNA kit (Quiagen, Hilden, Germany) according to the manufacturer’s instruction. All women were screened for the SNP at position 2039 (codon 680) of exon 10 of the FSHR gene as described earlier (19, 20). The SNP at position 260 in exon 2 of the DAZL gene was determined as described previously (21). Both assays were performed by the TaqMan allelic discrimination assay, using the ABI Prism 7000 sequence detection system (Applied Biosystems, Darmstadt, Germany).
In the present study, SNPs of the FSH receptor and of the DAZL gene were investigated and correlated to the timing of menarche and menopause.
MATERIALS AND METHODS Subjects The study protocol was approved by the institutional review board of the Academic Health Center of Siena. In a prospective study design, we enrolled 251 postmenopausal Italian women aged 43–85 years between January 2005 and November 2005. Approximately half of the study population (n ¼ 121) consisted of women seeking counseling for various reasons, mainly for postmenopausal disorders or risk assessment for malignancies, recruited at the Division of Obstetrics and Gynecology of the University of Siena. Written consent was obtained from all participating women. Age at menarche and menopause were available in all women, along with information concerning number of full-term pregnancies and miscarriages, history of recurrent abortion, age at the first delivery, smoking habits, and personal history of gynecological diseases. Patient inclusion criterion was physiological menopause. Women with a history of perimenopausal hormone therapy use were excluded. Further exclusion criteria Fertility and Sterility
Statistical Analysis Statistical analysis was performed with a commercially available statistical package (GraphPad Prism; GraphPad Software, San Diego, CA). Data are presented as medians and interquartile intervals, and group comparisons were performed by Kruskall-Wallis analysis of variance followed by Dunn’s test for multiple comparisons, or by Mann-Whitney test (two groups). P<.05 was considered statistically significant. Power and sample size calculations revealed that a minimum of 60 cases per group would be necessary to detect differences of 1 year in menarche age and 2 years in menopause age, with 80% statistical power and 95% confidence (22). RESULTS Menarche age, menopause age, and total years of fertility (difference between age of menopause and age of menarche) were evaluated in relation to the FSHR and DAZL genotype groups. The genotype distribution for FSHR followed Mendelian equilibrium, with 73 (29%) of the subjects being classified as Asn/Asn, 114 (45%) as Asn/Ser, and 64 (26%) as Ser/Ser. The median age at the time of the study was 63.0 years in the Asn/Asn group, 65.0 years in the Asn/Ser group, and 65.5 years in the Ser/Ser group, showing no significant difference between the three groups (P¼.959, Kruskal-Wallis analysis of variance). Depending on the FSH receptor genotype, a significant difference of menarche age was found (Fig. 1A). In detail, the median age of menarche was 13 years in the Asn/Asn 2265
FIGURE 1 Age of menarche (A), age of menopause (B), and duration of fertility (C) in 251 postmenopausal Italian women genotyped for FSH receptor gene polymorphism at codon 680. The box plots indicate the first and third quartiles, with the horizontal line representing the median; the error bars correspond to percentiles 10 and 90, and extreme values are represented by circles. Survival plots for the occurrence of menopause starting from the age of menarche do not show difference between the three evaluated phenotypes (D). The statistically significant differences indicated in (A) were obtained by Kruskal-Wallis analysis of variance and Dunn’s test.
A
70
p<0.01
Age of menopause (yr)
18
Age of menarche (yr)
B
p<0.05
16 14 12 10 8
Asn/Asn
Asn/Ser
60
50
40
30
Ser/Ser
Asn/Asn
C
Ser/Ser
D 50
1.0
Menopause (cumulative rate)
Length of fertile period (yr)
Asn/Ser
FSHR genotype
FSHR genotype
40
30
20
.8 .6 .4 Asn/Asn Asn/Ser Ser/Ser
.2 0.0
Asn/Asn
Asn/Ser
20
Ser/Ser
30
40
50
Time from menarche (yr)
FSHR genotype Zerbetto. FSH receptor and DAZL gene in menopause. Fertil Steril 2008.
group and 12 years in the Asn/Ser and Ser/Ser groups (Asn/ Asn vs. Asn/Ser, P<.01; Asn/Asn vs. Ser/Ser, P<.05, Dunn’s test). The median age at menopause was 50 years in the Asn/Asn and Asn/Ser groups and 51 years in the Ser/Ser/ group, a nonsignificant difference (P¼.132, Kruskal-Wallis analysis of variance). The median duration of fertility was 37 years in the Asn/Asn group, 38 years in the Asn/ Ser group, and 39 years in the Ser/Ser group, but this difference did not reach statistical significance (P¼.141). Accordingly, the analysis-of-survival curves depicted in Figure 1D show no difference in the occurrence of menopause starting from the age of menarche in the three different FSHR genotypes. Regarding the SNPs of DAZL, the distribution was 75% for the A/A genotype, 23% for A/G, and 2% for G/G. The A/G and G/G genotypes were considered together in the statistical anal2266
Zerbetto et al.
ysis. No significant correlation was found between the groups for menarche, menopause, and duration of fertility (Table 1). DISCUSSION The present study reports an association of FSHR gene polymorphism with the age at menarche but not with the age at natural menopause in a group of postmenopausal women. No correlation between the DAZL gene and the various parameters was observed. The timing of menarche and menopause are the cornerstones of a woman’s reproductive life and may influence the overall duration of her exposure to estrogen and then affect health in later life. In women with ovarian function the polymorphism at codon 680 of the FSHR is an important determinant of ovarian sensitivity to FSH (10). In particular, the
FSH receptor and DAZL gene in menopause
Vol. 90, No. 6, December 2008
TABLE 1 Age at menarche, age at menopause, and duration of fertility according to DAZL genotypes at nucleotide position 260. Genotype
A/A
A/G D G/G
P
No. of cases 188 63 — Frequency (%) 75 25 — Age at 12.5 13.0 .75 menarche (y) (12.014.0) (12.014.0) Age at 50.0 50.0 .97 menopause (y) (48.053.0) (48.053.0) Duration 38.0 38.0 .94 of fertility (y) (35.040.0) (35.040.0) Note: Data are expressed as median (interquartile range) and compared by Mann-Whitney test. Zerbetto. FSH receptor and DAZL gene in menopause. Fertil Steril 2008.
Ser680 receptor variant seems to be less ‘‘sensitive’’ to FSH stimulation, and women homozygous for this variant have significantly higher serum FSH levels in the follicular phase of the menstrual cycle than women with the Asn680 isoform. In addition, it was recently demonstrated that the SNP at codon 680 is a major determinant of menstrual cycle length and hormonal dynamics and that FSH-dependent E2 production is different in women undergoing controlled ovarian hyperstimulation depending on this SNP. Such different responsiveness to FSH is not evident in men, both with normal or impaired spermatogenesis (9, 23). This sex-related difference is striking, and it is difficult to understand why no functional consequence of the exon 10 SNP is observed in men. In the present study we have found that women with the Asn/Asn variant of FSHR polymorphism at codon 680 (Asn680 isoform) had their menarche 1 year later than the other genotype groups. The correlation between the age at menarche and FSHR polymorphism in healthy postmenopausal women suggests a possible impact of FSH receptor gene in the modulation of the onset of puberty. We have previously shown that women with the Asn/Asn genotype have lower serum FSH concentrations despite the same inhibin and E2 levels of the remaining groups (12). Asn/Asn women also require less exogenous FSH during controlled ovarian stimulation for infertility treatment, indicating that this receptor variant is more sensitive to FSH (10). The finding of later menarche in a group with greater FSHR sensitivity and totally preserved ovarian function suggests that these women might have an increased hypothalamic sensitivity to FSH short feedback loop, thus delaying the start of pubertal GnRH pulsatility. This hypothesis, however, remains speculative and should be tested experimentally. In the present study the distribution of the DAZL SNP 260 was not associated with the onset of menarche or with the age Fertility and Sterility
at menopause. The timing of menarche is regulated by a variety of environmental and genetic factors. Family and twin studies have indicated that the genetic contribution may be more important than environmental effects, because 53%–74% of the variation in age of menarche can be attributed to genetic factors (24, 25). However, the specific genes involved in this event are not yet well defined. Other DAZL polymorphisms have been detected with higher frequency in women with premature ovarian failure and have been associated with an earlier age at ovarian failure in this pathologic group, but not in women reporting menopause after age 40 years (17). This fact, together with the present finding that the DAZL SNP 260 is not related to age at menopause or to duration of fertility in a population without premature ovarian failure suggests that DAZL products, although critical for ovarian development (16), do not play a central role in defining subtle differences of ovarian reserve in normal individuals. Nevertheless, testing candidate gene markers associated with ovarian reserve and menopause may provide clinical tools not only to reproductive counseling but also for healthpromoting actions. An early onset of menopause is related to higher risks of cardiovascular diseases (26), osteoporosis (27), and ovarian cancer (28), increasing the risk of mortality. Delayed menopause, however, increases the risk of endometrial and breast cancer (29). Several environmental factors, such as cigarette smoking, nutrition, sociodemographic factors, and reproductive characteristics, have been proposed as risk factors for the early onset of menopause. These recognized trends, as well as the fact that menopause is preceded by a period of reduced fertility, further increase the interest relevance of factors predicting the timing of natural menopause (30, 31). To find genetic markers of early or delayed menopause may therefore have important clinical usefulness for the screening and prevention of degenerative diseases. Because the present study was based on patient report of age at menarche and menopause, its findings should be confirmed by prospective evaluation of pubertal girls and perimenopausal women to rule out the possibility of recall bias, although systematic bias is unlikely to explain differences between the genotype groups compared in the study, because patients were asymptomatic and unaware of their own genotype. Also, any extrapolation of our findings must consider that the study was confined to women of Caucasian/Italian ethnicity. In conclusion, our results showed that genotype of FSH receptor or SNP of DAZL are not predictive of the age at natural menopause and duration of fertility in women. The presence of the Asn680/Asn680 genotype is associated with a slightly delayed age at menarche.
REFERENCES 1. Meyer JM, Eaves LJ, Heath AC, Martin NG. Estimating genetic influences on the age-at-menarche: a survival analysis approach. Am J Med Genet 1991;39:148–54.
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2. Kaprio J, Rimpela A, Winter T, Viken RJ, Rimpela M, Rose RJ. Common genetic influences on BMI and age at menarche. Hum Biol 1995;67: 739–53. 3. Carr BR. Disorders of the ovaries and female reproductive tract. In: Wilson JD, Forester DW, Kronenberg HM, Carsen PR, eds. Williams textbook of endocrinology. 9th ed. New York: W.B. Saunders, 1998:751–818. 4. Tempfer CB, Schneeberger C, Huber JC. Applications of polymorphisms and pharmacogenomics in obstetrics and gynecology. Pharmacogenomics 2004;5:57–65. 5. Gorai I, Tanaka K, Inada M, Morinaga H, Uchiyama Y, Kikuchi R, et al. Estrogen-metabolizing gene polymorphisms, but not estrogen receptoralpha gene polymorphisms, are associated with the onset of menarche in healthy postmenopausal Japanese women. J Clin Endocrinol Metab 2003;88:799–803. 6. Weel AE, Uitterlinden AG, Westerndop IC, Burger H, Schuit SC, Hofman A, et al. Estrogen receptor polymorphism predicts the onset of natural and surgical menopause. J Clin Endocrinol Metab 1999;84: 3146–50. 7. Zheng W, Magid MS, Kramer EE, Chen YT. Follicle-stimulating hormone receptor is expressed in human ovarian surface epithelium and fallopian tube. Am J Pathol 1996;148:47–53. 8. Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophisiology. Endocr Rev 1997;18:739–73. 9. Simoni M, Gromoll J, H€oppner W, Kamischke A, Krafft T, St€ahle D, et al. Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. J Clin Endocrinol Metab 1999;84:751–5. 10. Perez-Mayorga M, Gromoll J, Behre HM, Gassner C, Nieschlag E, Simoni M. Ovarian response to follicle-stimulating hormone (FSH) stimulating depends on the FSH receptor genotype. J Clin Endocrinol Metab 2000;85:3365–9. 11. Behre HM, Greb RR, Mempel A, Sonntag B, Kiesel L, Kaltwasser P, et al. Significance of a common single nucleotide polymorphism in exon 10 of the follicle-stimulating hormone (FSH) receptor gene for the ovarian response to FSH: a pharmacogenetic approach to controlled ovarian hyperstimulation. Pharmacogenomics 2005;15:451–6. 12. Greb RR, Grieshaber K, Gromoll J, Sonntag B, Nieschlag E, Kiesel L, et al. A common single nucleotide polymorphism in exon 10 of the human follicle stimulating hormone receptor is a major determinant of length and hormonal dynamics of the menstrual cycle. J Clin Endocrinol Metab 2005;90:4866–72. 13. Reijo RA, Dorfman DM, Slee R, Renshaw AA, Loughlin KR, Cooke H, et al. DAZL family proteins exists throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice. Biol Reprod 2000;63:1490–4. 14. Xu EY, Moore FL, Reijo RP. A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans. Proc Natl Acad Sci U S A 2001;98:7414–9.
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15. Lin YM, Chen CW, Sun HS, Tsai SJ, Lin JS, Kuo PL. Presence of DAZL transcript and protein in mature human spermatozoa. Fertil Steril 2002;77:626–9. 16. McNeilly JR, Saunders PT, Taggart M, Cranfield M, Cooke HJ, McNeilly AS. Loss of oocytes in Dazl knockout mice results in maintained ovarian steroidogenic function but altered gonadotropin secretion in adult animals. Endocrinology 2000;141:4284–94. 17. Tung JY, Rosen MP, Nelson LM, Turek PJ, Witte JS, Cramer DW, et al. Variants in Deleted in AZoospermia-Like (DAZL) are correlated with reproductive parameters in men and women. Hum Genet 2006;118: 730–40. 18. Teng YN, Lin YM, Lin HM, Tsao SY, Hsu CC, Lin SJ, et al. Association of a single nucleotide polymorphism of the DAZL gene with susceptibility to spermatogenic failure. J Clin Endocrinol Metab 2002;87:5258–64. 19. Ahda Y, Gromoll J, Wunsch A, Asatiani K, Zitzmann M, Nieschlag E, et al. FSH receptor gene haplotype distribution in normozoospermic and azoospermic men. J Androl 2005;26:494–9. 20. Wunsch A, Ahda Y, Banaz-Yasar F, Sonntag B, Nieschlag E, Simoni M, et al. Single nucleotide polymorphism in the promoter region influence the expression of the human follicle-stimulating hormone receptor. Fertil Steril 2005;84:446–53. 21. Tschanter P, Kostova E, Lutjens CM, Cooper TG, Nieschlag E, Gromoll J. No association of the A260G and A386g DAZL single nucleotide polymorphisms with male infertility in a Caucasian population. Hum Reprod 2004;19:2771–6. 22. Machin D, Campbell M, Fayers P, Pinol A. Sample size tables for clinical studies. 2nd ed. Malden, MA: Blackwell Science, 1997. 23. Asatiani K, Gromoll J, Eckardstein SV, Zitzmann M, Nieshlag E, Simoni M. Distribution and function of FSH receptor genetic variants in normal men. Andrologia 2002;34:172–6. 24. Sharma JC. The genetic contribution to pubertal growth and development studied by longitudinal growth data on twins. Ann Hum Biol 1983;10: 163–71. 25. Treloar SA, Martin NG. Age at menarche as a fitness trait. Nonadditive genetic variance detected in a large twin sample. Am J Hum Genet 1990;47:137–48. 26. Van der Schouwn Yt, Van der Graaf Y, Steyerberg EW, Eijkemans MJC, Banga JD. Age at menopause as a risk factor for cardiovascular mortality. Lancet 1996;347:714–8. 27. Falch JA, Sandvik L. Perimenopausal appendicular bone loss. A 10 year prospective study. Bone 1990;11:425–8. 28. Cramer DW. Epidemiology aspects of early menopause and ovarian cancer. Ann N Y Acad Sci 1990;592:363–75. 29. Swan SH. Hormone replacement therapy and the risk of reproductive cancer. J Psychosom Obstet Gynecol 1997;18:165–74. 30. Kato I, Toniolo P, Akhmedkhanova A, Koenig KL, Shore R, ZeleniuchJacquotte A. Prospective study of factors influencing the onset of natural menopause. J Clin Epidemiol 1998;51:1271–6. 31. Harlow BL, Signorello LB. Factors associated with early menopause. Maturitas 2000;35:3–9.
FSH receptor and DAZL gene in menopause
Vol. 90, No. 6, December 2008
Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy; and b Institute of Reproductive Medicine, University Hospital, M€unster, Germany
Objective: To evaluate whether single nucleotide FSH receptor and DAZL gene polymorphisms are associated with menarche and menopause timing. Design: Prospective study. Setting: Siena and M€unster Universities. Patient(s): Physiologically menopausal women. Intervention(s): The presence of FSH receptor or DAZL gene polymorphisms was investigated. Blood samples were collected and polymorphisms evaluated in extracted genomic DNA. Main Outcome Measure(s): Menarche age, menopausal age, and total years of fertility were evaluated on the basis of FSH receptor (genotypes Asn/Asn, Asn/Ser, and Ser/Ser at codon 680) and DAZL gene. Result(s): The median age of menarche was 13 years in the Asn/Asn group and 12 years in the Asn/Ser and Ser/Ser groups. The median age at menopause was 50 years in the Asn/Asn and Asn/Ser groups and 51 years in the Ser/Ser group. The length of the fertile period was 37 years in the Asn/Asn group, 38 years in the Asn/Ser group, and 39 years in the Ser/Ser group. Regarding the DAZL polymorphism, A/A, A/G, and G/G had the same age at menarche, age at menopause, and length of fertile period. Conclusion(s): We found that genotype of FSH receptor or SNP of DAZL do not predict the age at natural menopause and duration of fertility in women. The presence of Asn680/Asn680 genotype is associated with a slightly delayed age at menarche. (Fertil Steril 2008;90:2264–8. 2008 by American Society for Reproductive Medicine.) Key Words: FSH receptor, DAZL, menarche, menopause, SNP
Genetic influences have been shown to contribute greatly to the timing of puberty (1, 2) and of menopause. Menarche and menopause depend on the interaction of the hypothalamic–pituitary–ovarian axis with the female reproductive system and other endocrine organs (3). Menarche and menopause are characterized, respectively, by a physiological increase and decrease of ovarian hormone production, causing typical somatic and/or psychological alterations. In the last decade it has been shown that single nucleotide polymorphisms (SNPs) are responsible for the interindividual variation of numerous physiological functions, among them blood pressure, drug metabolism, blood clotting, and cardiovascular dysfunction (4). Preliminary studies Received June 29, 2007; revised October 18, 2007; accepted October 25, 2007. Supported by grants from the Italian Ministry of University and Scientific Research and the University of Siena. The funding source had no role in the study design, data collection, data interpretation, data analysis, or writing of the report. Reprint requests: Felice Petraglia, M.D., Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena Policlinico ‘‘Le Scotte’’ Viale Bracci, 53100 Siena, Italy (FAX: 39-0577-233-454; E-mail: [email protected]).
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have shown a correlation between SNPs in genes involved in estrogen metabolism and the timing of menarche (5), and between SNP in the estrogen receptor gene and the timing of menopause (5, 6). Follicle-stimulating hormone is a key hormone in the modulation of the hypothalamic–pituitary–ovarian axis, and its receptors are expressed in women mainly in ovarian granulosa cells (7). The FSH receptor (FSHR) belongs to the family of G protein-coupled receptors inducing signal transduction by the protein kinase A/cyclic adenosine monophosphate pathway (8). Apart from rare mutations, two common SNPs were identified in exon 10 of the FSHR gene at nucleotide positions 919 and 2039, respectively. The first SNP is located in the extracellular domain at codon position 307 (National Center for Biotechnology Information [NCBI] reference SNP ID: rs6165), which can be occupied either by alanine (Ala) or by threonine (Thr). The second SNP is located in the intracellular domain at codon position 680 and has either asparagine (Asn) or serine (Ser) (NCBI reference SNP ID: rs6166). The two SNPs are mostly in linkage disequilibrium in the Caucasian population, resulting in two discrete allelic variants, Thr307-Asn680 and Ala307-Ser680 (9), the latter
Fertility and Sterility Vol. 90, No. 6, December 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.036
representing approximately 40% of FSH receptor alleles worldwide.
were surgical menopause, known gynecological diseases, and premature ovarian failure.
The FSHR 680 polymorphism has been investigated in women undergoing controlled ovarian hyperstimulation (10, 11), in whom FSHR isoforms are involved in determining the biological effects of the hormone and may help to define the optimal FSH starting dose. Recently, different kinetics of hormonal secretion and longer duration of menstrual cycles in women with the Ser680/Ser680 genotype have been shown (12), raising the question of whether this FSHR polymorphism has any role in determining the duration of fertility and the age at which menarche and menopause occur.
The remaining subjects (n ¼ 130) were women undergoing ambulatory diagnostic procedures at the clinical chemistry laboratory of the University of Siena who volunteered to donate a blood sample for the study. These women answered a questionnaire asking for age at menarche and physiological menopause. Personal identifiers were removed from all samples. Menarche was defined as age at first menstrual period (as recalled by the study participants); natural menopause was defined as age of the last period without having further menstrual bleeding for at least 12 consecutive months.
In this field, a family of RNA binding protein, the DAZ (deleted in azoospermia) gene family, has been identified. It consists of two autosomal genes, namely BOULE and DAZL (DAZ-like), and the Y-chromosomal DAZ gene cluster. These genes are expressed exclusively in prenatal and/or postnatal germ cells and are assumed to be master regulators of germline expression (13–15). Studies in DAZL knockout mice (16) showed the complete absence of oocytes, suggesting that DAZL is involved in oocyte maturation and, possibly, in follicular exhaustion. Some DAZL polymorphisms have being associated with premature ovarian failure, although not with the age of physiological menopause (17). Of particular interest is an SNP located at the DAZL nucleotide position 260 (exon 2; A/G change), just upstream of the RNA binding domain, which leads to a threonine-to-alanine change at amino acid position 12 with possible impact on germ cell reserve (18). Whether this particular SNP is related to the duration of the fertile period in women is still unknown.
DNA Isolation and Analysis Genomic DNA was obtained from peripheral blood leukocytes using the Flexi Gene DNA kit (Quiagen, Hilden, Germany) according to the manufacturer’s instruction. All women were screened for the SNP at position 2039 (codon 680) of exon 10 of the FSHR gene as described earlier (19, 20). The SNP at position 260 in exon 2 of the DAZL gene was determined as described previously (21). Both assays were performed by the TaqMan allelic discrimination assay, using the ABI Prism 7000 sequence detection system (Applied Biosystems, Darmstadt, Germany).
In the present study, SNPs of the FSH receptor and of the DAZL gene were investigated and correlated to the timing of menarche and menopause.
MATERIALS AND METHODS Subjects The study protocol was approved by the institutional review board of the Academic Health Center of Siena. In a prospective study design, we enrolled 251 postmenopausal Italian women aged 43–85 years between January 2005 and November 2005. Approximately half of the study population (n ¼ 121) consisted of women seeking counseling for various reasons, mainly for postmenopausal disorders or risk assessment for malignancies, recruited at the Division of Obstetrics and Gynecology of the University of Siena. Written consent was obtained from all participating women. Age at menarche and menopause were available in all women, along with information concerning number of full-term pregnancies and miscarriages, history of recurrent abortion, age at the first delivery, smoking habits, and personal history of gynecological diseases. Patient inclusion criterion was physiological menopause. Women with a history of perimenopausal hormone therapy use were excluded. Further exclusion criteria Fertility and Sterility
Statistical Analysis Statistical analysis was performed with a commercially available statistical package (GraphPad Prism; GraphPad Software, San Diego, CA). Data are presented as medians and interquartile intervals, and group comparisons were performed by Kruskall-Wallis analysis of variance followed by Dunn’s test for multiple comparisons, or by Mann-Whitney test (two groups). P<.05 was considered statistically significant. Power and sample size calculations revealed that a minimum of 60 cases per group would be necessary to detect differences of 1 year in menarche age and 2 years in menopause age, with 80% statistical power and 95% confidence (22). RESULTS Menarche age, menopause age, and total years of fertility (difference between age of menopause and age of menarche) were evaluated in relation to the FSHR and DAZL genotype groups. The genotype distribution for FSHR followed Mendelian equilibrium, with 73 (29%) of the subjects being classified as Asn/Asn, 114 (45%) as Asn/Ser, and 64 (26%) as Ser/Ser. The median age at the time of the study was 63.0 years in the Asn/Asn group, 65.0 years in the Asn/Ser group, and 65.5 years in the Ser/Ser group, showing no significant difference between the three groups (P¼.959, Kruskal-Wallis analysis of variance). Depending on the FSH receptor genotype, a significant difference of menarche age was found (Fig. 1A). In detail, the median age of menarche was 13 years in the Asn/Asn 2265
FIGURE 1 Age of menarche (A), age of menopause (B), and duration of fertility (C) in 251 postmenopausal Italian women genotyped for FSH receptor gene polymorphism at codon 680. The box plots indicate the first and third quartiles, with the horizontal line representing the median; the error bars correspond to percentiles 10 and 90, and extreme values are represented by circles. Survival plots for the occurrence of menopause starting from the age of menarche do not show difference between the three evaluated phenotypes (D). The statistically significant differences indicated in (A) were obtained by Kruskal-Wallis analysis of variance and Dunn’s test.
A
70
p<0.01
Age of menopause (yr)
18
Age of menarche (yr)
B
p<0.05
16 14 12 10 8
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Asn/Ser
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50
40
30
Ser/Ser
Asn/Asn
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Ser/Ser
D 50
1.0
Menopause (cumulative rate)
Length of fertile period (yr)
Asn/Ser
FSHR genotype
FSHR genotype
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.8 .6 .4 Asn/Asn Asn/Ser Ser/Ser
.2 0.0
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FSHR genotype Zerbetto. FSH receptor and DAZL gene in menopause. Fertil Steril 2008.
group and 12 years in the Asn/Ser and Ser/Ser groups (Asn/ Asn vs. Asn/Ser, P<.01; Asn/Asn vs. Ser/Ser, P<.05, Dunn’s test). The median age at menopause was 50 years in the Asn/Asn and Asn/Ser groups and 51 years in the Ser/Ser/ group, a nonsignificant difference (P¼.132, Kruskal-Wallis analysis of variance). The median duration of fertility was 37 years in the Asn/Asn group, 38 years in the Asn/ Ser group, and 39 years in the Ser/Ser group, but this difference did not reach statistical significance (P¼.141). Accordingly, the analysis-of-survival curves depicted in Figure 1D show no difference in the occurrence of menopause starting from the age of menarche in the three different FSHR genotypes. Regarding the SNPs of DAZL, the distribution was 75% for the A/A genotype, 23% for A/G, and 2% for G/G. The A/G and G/G genotypes were considered together in the statistical anal2266
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ysis. No significant correlation was found between the groups for menarche, menopause, and duration of fertility (Table 1). DISCUSSION The present study reports an association of FSHR gene polymorphism with the age at menarche but not with the age at natural menopause in a group of postmenopausal women. No correlation between the DAZL gene and the various parameters was observed. The timing of menarche and menopause are the cornerstones of a woman’s reproductive life and may influence the overall duration of her exposure to estrogen and then affect health in later life. In women with ovarian function the polymorphism at codon 680 of the FSHR is an important determinant of ovarian sensitivity to FSH (10). In particular, the
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TABLE 1 Age at menarche, age at menopause, and duration of fertility according to DAZL genotypes at nucleotide position 260. Genotype
A/A
A/G D G/G
P
No. of cases 188 63 — Frequency (%) 75 25 — Age at 12.5 13.0 .75 menarche (y) (12.014.0) (12.014.0) Age at 50.0 50.0 .97 menopause (y) (48.053.0) (48.053.0) Duration 38.0 38.0 .94 of fertility (y) (35.040.0) (35.040.0) Note: Data are expressed as median (interquartile range) and compared by Mann-Whitney test. Zerbetto. FSH receptor and DAZL gene in menopause. Fertil Steril 2008.
Ser680 receptor variant seems to be less ‘‘sensitive’’ to FSH stimulation, and women homozygous for this variant have significantly higher serum FSH levels in the follicular phase of the menstrual cycle than women with the Asn680 isoform. In addition, it was recently demonstrated that the SNP at codon 680 is a major determinant of menstrual cycle length and hormonal dynamics and that FSH-dependent E2 production is different in women undergoing controlled ovarian hyperstimulation depending on this SNP. Such different responsiveness to FSH is not evident in men, both with normal or impaired spermatogenesis (9, 23). This sex-related difference is striking, and it is difficult to understand why no functional consequence of the exon 10 SNP is observed in men. In the present study we have found that women with the Asn/Asn variant of FSHR polymorphism at codon 680 (Asn680 isoform) had their menarche 1 year later than the other genotype groups. The correlation between the age at menarche and FSHR polymorphism in healthy postmenopausal women suggests a possible impact of FSH receptor gene in the modulation of the onset of puberty. We have previously shown that women with the Asn/Asn genotype have lower serum FSH concentrations despite the same inhibin and E2 levels of the remaining groups (12). Asn/Asn women also require less exogenous FSH during controlled ovarian stimulation for infertility treatment, indicating that this receptor variant is more sensitive to FSH (10). The finding of later menarche in a group with greater FSHR sensitivity and totally preserved ovarian function suggests that these women might have an increased hypothalamic sensitivity to FSH short feedback loop, thus delaying the start of pubertal GnRH pulsatility. This hypothesis, however, remains speculative and should be tested experimentally. In the present study the distribution of the DAZL SNP 260 was not associated with the onset of menarche or with the age Fertility and Sterility
at menopause. The timing of menarche is regulated by a variety of environmental and genetic factors. Family and twin studies have indicated that the genetic contribution may be more important than environmental effects, because 53%–74% of the variation in age of menarche can be attributed to genetic factors (24, 25). However, the specific genes involved in this event are not yet well defined. Other DAZL polymorphisms have been detected with higher frequency in women with premature ovarian failure and have been associated with an earlier age at ovarian failure in this pathologic group, but not in women reporting menopause after age 40 years (17). This fact, together with the present finding that the DAZL SNP 260 is not related to age at menopause or to duration of fertility in a population without premature ovarian failure suggests that DAZL products, although critical for ovarian development (16), do not play a central role in defining subtle differences of ovarian reserve in normal individuals. Nevertheless, testing candidate gene markers associated with ovarian reserve and menopause may provide clinical tools not only to reproductive counseling but also for healthpromoting actions. An early onset of menopause is related to higher risks of cardiovascular diseases (26), osteoporosis (27), and ovarian cancer (28), increasing the risk of mortality. Delayed menopause, however, increases the risk of endometrial and breast cancer (29). Several environmental factors, such as cigarette smoking, nutrition, sociodemographic factors, and reproductive characteristics, have been proposed as risk factors for the early onset of menopause. These recognized trends, as well as the fact that menopause is preceded by a period of reduced fertility, further increase the interest relevance of factors predicting the timing of natural menopause (30, 31). To find genetic markers of early or delayed menopause may therefore have important clinical usefulness for the screening and prevention of degenerative diseases. Because the present study was based on patient report of age at menarche and menopause, its findings should be confirmed by prospective evaluation of pubertal girls and perimenopausal women to rule out the possibility of recall bias, although systematic bias is unlikely to explain differences between the genotype groups compared in the study, because patients were asymptomatic and unaware of their own genotype. Also, any extrapolation of our findings must consider that the study was confined to women of Caucasian/Italian ethnicity. In conclusion, our results showed that genotype of FSH receptor or SNP of DAZL are not predictive of the age at natural menopause and duration of fertility in women. The presence of the Asn680/Asn680 genotype is associated with a slightly delayed age at menarche.
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