- Email: [email protected]
G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve
G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR. (Fertil Steril 2010;94:2490–2. 2010 by American Society for Reproductive Medicine.) Key Words: Diminished ovarian reserve, GDF-9, ovarian stimulation, polymorphism
Ovarian reserve, the term used to describe the capacity of the ovary to provide oocytes, reflects the quantity and quality of oocytes within the ovary at any given time (1). Diminished ovarian reserve (DOR), a decreased number of oocytes and a decrease in oocyte quality in women of reproductive age, is commonly associated with a poor response to ovarian stimulation during an in vitro fertilization (IVF) cycle (2, 3). The presence of DOR may also portend the occurrence of premature ovarian aging and Ting-Ting Wang, M.D.a,b Yan-Ting Wu, Ph.D.a,b Min-Yue Dong, Ph.D.a,b Jian-Zhong Sheng, Ph.D.c Peter C.K. Leung, Ph.D.d He-Feng Huang, M.D.a,b a Department of Reproductive Endocrinology, Zhejiang Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China b Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Hangzhou, People’s Republic of China c Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China d Department of Obstetrics and Gynecology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Received November 5, 2009; revised March 26, 2010; accepted March 29, 2010; published online May 7, 2010. T-T.W. has nothing to disclose. Y-T.W. has nothing to disclose. M-Y.D. has nothing to disclose. J-Z.S. has nothing to disclose. P.C.K.L. has nothing to disclose. H-F.H. has nothing to disclose. Supported by the China-Canada Joint Health Research Initiative Grants Program (30711120577), the National Natural Science Foundation of China (30900517), and an Innovation Award for Graduate Students of Zhejiang Province (YK2008008). Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Zhejiang Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, People’s Republic of China (FAX: 86-571-8706-1878; E-mail: [email protected]).
2490
menopause (4). Factors such as GDF-9, BMP-15, AMH, GPR3, and NOBOX are known to influence the growth and depletion rate of follicles (5, 6). The genetic variants of these factors are associated with abnormal follicular loss and might result in premature ovarian failure (POF) (6). Growth differential factor-9 (GDF-9), a member of the transforming growth factor-b superfamily, is an oocyte-derived factor that is preferentially expressed in the oocytes of humans and mice. Because GDF-9 plays a pivotal role during early folliculogenesis, deletion of GDF-9 in the mouse causes follicular arrest at the primary stage and infertility (7). Also, GDF-9 stimulates granulosa cell proliferation (8) and cumulus cell expansion (9), inhibits follicular apoptosis (10), and enhances oocyte and embryo development (11, 12). Recently, several genetic variants of GDF-9 have been identified, and their correlation with POF has been noted, suggesting that these variants contribute to aberrant follicular development and oocyte loss (13–15). We investigated the possible correlation of GDF-9 polymorphisms with DOR and evaluated the contribution of GDF-9 polymorphisms to poor IVF outcomes in women with DOR. A total of 103 women with DOR were recruited consecutively during their IVF treatment in the Reproductive Center of Zhejiang Women’s Hospital, Zhejiang University. The diagnosis of DOR was based on a (day-3) basal antral follicle count <5 combined a with basal follicle-stimulating hormone (FSH) level >10 IU/L and/or basal estradiol (E2) >80 pg/mL, as described previously elsewhere (16). All DOR diagnoses were made after at least two cycles. Other factors affecting ovarian functions were excluded: ovarian surgery, polycystic ovarian syndrome or polycystic ovaries, endometriosis, or body mass index >25. None of the women had taken hormone treatment or had endocrine disorders. The control group consisted of 123 age-matched women with normal ovarian reserve and infertility due to tubular or unexplained causes. All participants were Han Chinese from the southeast of China and had no genetic relationships. Informed consent was given by all participants for the use of their blood samples, and the ethics committee of Zhejiang University reviewed and approved this study.
Fertility and Sterility Vol. 94, No. 6, November 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.03.070
Genomic DNA was extracted from leukocytes in the peripheral blood of the women using the QiaAmp DNA Blood kit (Qiagen, Hilden, Germany). The coding regions of GDF-9 were amplified using a polymerase chain reaction (PCR) protocol (detailed information concerning the conditions is available upon request). The PCR products were sequenced using the Big Dye terminator sequencing protocol with a Big-Dye Terminator kit (Applied Biosystems, Foster City, CA) and were analyzed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Hardy-Weinberg testing for each polymorphism used Haploview version 3.2 (18). Comparisons of the distributions of genotype frequencies were performed using Fisher’s exact test or the chi-square test. Differences in clinical parameters among genotypes were tested using Student’s t-test or one-way analysis of variance for continuous variables. Categorical parameters were analyzed using Fisher’s exact test. The statistical analyses were performed with Statistical Package for the Social Sciences software version 15.0 (SPSS, Inc., Chicago, IL). P<.05 was considered statistically significant. Three variants of the GDF-9 gene, G169A, C447T (rs254286), and G546A (rs10491279), were identified by DNA sequencing. Of these, C447T and G546A are in the single nucleotide polymorphism (SNP) database, and G169A is not yet included. All these polymorphisms conformed to the Hardy-Weinberg equilibrium. A statistically significant correlation was found between the GDF-9 G546A genotype and ovarian reserve status. Thirty-four of 106 women with DOR (32.1%; CI, 23.3–41.9%) had the GA/ AA genotype compared with 24 of 123 women with normal ovarian reserve (19.5%; CI, 12.9–27.6%; P<.05).
Wang. Correspondence. Fertil Steril 2010.
Note: Values in parentheses are 95% confidence interval. MII ¼ metaphase II; rFSH ¼ recombinant follicle-stimulating hormone. a Values are mean standard deviation and compared with Student’s t-test. b Values are compared with Fisher’s exact test.
< .001 < .001 .032 < .001 < .001 .022 3037 967.2 (2,670–3,374) 64.7 (46.5–80.3) 2.41 2.33 (1.60–3.222) 23.9 (17.5–31.3) 5.3 (0.65–17.7) 6.7 (0.77–20.7) .366 .160 .436 .064 .492 .637 2371 996.6 (1,950–27,926) 33.3 (15.6–55.4) 5.29 2.03 (4.43–6.15) 71.6 (22.1–35.3) 64.7 (56.2–72.7) 36.4 (17.2–59.3) rFSH dose used (IU)a Poor ovarian response (%)b No. of MII oocytesa Fertilization rate (%)b Good quality embryos (%)b Pregnancy rate (%)b
2364 912.7 (2,182–2,546) 18.2 (11.4–27.3) 5.77 1.93 (5.39–6.16) 77.8 (75.1–80.4) 67.9 (64.5–71.2) 43 (32.8–53.8)
2,700 833.3 (2,494–2,906) 23.6 (14.4–35.5) 4.81 2.79 (4.15–5.47) 73.4 (69.4–77.2) 62.2 (57.1–67.1) 33.3 (16.4–35.3)
P value GA/AA P value GA/AA IVF outcome
GG
GG
Diminished ovarian reserve (n [ 106) Normal ovarian reserve (n [ 123)
Correlation between GDF9 G546A genotypes and in vitro fertilization outcomes in the diminished ovarian reserve patients and controls.
TABLE 1
Fertility and Sterility
The standard long down-regulation protocol of controlled ovarian stimulation (COH) was performed in all subjects as described before (17). Poor ovarian response was defined as the presence of %3 follicles with diameters of R14 mm on the day of oocyte retrieval or cycle cancellation. The presence of two pronuclei was considered to indicate fertilization. Embryos with R6 blastomeres and <20% fragmentation were considered quality embryos. Embryo transfer was performed on day 3, and a maximum of two embryos were placed in each woman. A clinical pregnancy was defined as the presence of fetal cardiac activity beyond 8 weeks of gestation.
Furthermore, we examined the correlations between polymorphisms and IVF outcomes in women with DOR or normal ovarian reserve (Table 1). In women with DOR, the carriers of the GDF-9 546A allele (GA/AA genotype) had a higher prevalence of poor ovarian response. Although these women were given more than the routine dose of recombinant FSH in controlled ovarian stimulation, they still had fewer metaphase II oocytes, a lower fertilization rate, fewer good quality embryos, and a lower pregnancy rate. In contrast, in controls, these parameters in women with the GA/ AA genotype did not differ from those with the GG genotype. However, no correlation between GDF-9 G169A or C447T and IVF outcomes was detected (data not shown). Although animal models and in vitro studies have revealed the role of GDF-9 in regulating follicular development (19), little is known about its role in human ovarian function. Our study demonstrated a strong correlation between GDF-9 G546A polymorphism in women with DOR and poor IVF outcomes, indicating that GDF9 plays an important role in determining ovarian reserve status and functions. Our findings also raise the intriguing possibility that this
2491
genetic variation of GDF-9 is responsible for the high frequency of suboptimal ovarian stimulation outcomes during IVF cycles in women with DOR. More importantly, we noted that, even though the women with the GA/AA genotype and DOR were treated with much higher doses of recombinant FSH, they still presented few good quality embryos and had a low pregnancy rate (5.3% and 6.7%), suggesting that increasing the dose of recombinant FSH is not effective in improving ovarian response and IVF outcomes in women with DOR who carry the GDF-9 546A allele. We did not detect other the GDF-9 variants such as A199C (K67E), G646A (V216M), C307T (P103S), or C557A (S186Y) that had been found in women with POF (13–15). This discrepancy may be due to our focus on patients with DOR rather than with POF. The mechanisms underlying the correlation between GDF-9 G546A polymorphism and DOR are
still unclear. Because this polymorphism is silent, there is a possibility that it has strong linkage disequilibrium with some causative sequence variants that may alter the function of GDF-9 by influencing its messenger RNA (mRNA) splicing or protein processing. Our study is the first demonstration of the role of GDF-9 genetic variants in ovarian reserve and IVF outcomes. Women with DOR and the GDF-9 G546A polymorphism showed poor IVF outcomes. These results show that the GDF-9 gene variant(s), at least in part, may be related to the poor IVF outcomes in women with DOR. Further studies are needed to clarify the mechanisms involved in the effects of these polymorphisms on ovarian functions. Acknowledgments: The authors thank Dr. Iain Bruce for reviewing and editing the manuscript, and the members of the Department of Reproductive Endocrinology in the Women’s Hospital, Zhejiang University, for their constant assistance throughout the study.
REFERENCES 1. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12:685–718. 2. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT Jr. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001;76:666–9. 3. te Velde ER, Pearson PL. The variability of female reproductive ageing. Hum Reprod Update 2002;8: 141–54. 4. Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003;18:527–33. 5. Simoni M, Tempfer CB, Destenaves B, Fauser BC. Functional genetic polymorphisms and female reproductive disorders: Part I: Polycystic ovary syndrome and ovarian response. Hum Reprod Update 2008;14:459–84. 6. Broekmans FJ, Soules MR, Fauser BC. Ovarian ageing: mechanisms and clinical consequences. Endocr Rev 2009;30:465–93. 7. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor9 is required during early ovarian folliculogenesis. Nature 1996;383:531–5.
2492
Wang et al.
Correspondence
8. Vitt UA, Hayashi M, Klein C, Hsueh AJ. Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles. Biol Reprod 2000;62: 370–7. 9. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, et al. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 2001;15:854–66. 10. Orisaka M, Orisaka S, Jiang JY, Craig J, Wang Y, Kotsuji F, et al. Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage. Mol Endocrinol 2006;20:2456–68. 11. Hussein TS, Thompson JG, Gilchrist RB. Oocytesecreted factors enhance oocyte developmental competence. Dev Biol 2006;296:514–21. 12. Yeo CX, Gilchrist RB, Thompson JG, Lane M. Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice. Hum Reprod 2008;23:67–73. 13. Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, et al. Mutations and
14.
15.
16.
17.
18.
19.
sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Eur J Endocrinol 2006;154:739–44. Chand AL, Ponnampalam AP, Harris SE, Winship IM, Shelling AN. Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure. Fertil Steril 2006;86: 1009–12. Kovanci E, Rohozinski J, Simpson JL, Heard MJ, Bishop CE, Carson SA. Growth differentiating factor-9 mutations may be associated with premature ovarian failure. Fertil Steril 2007;87:143–6. Sills ES, Alper MM, Walsh AP. Ovarian reserve screening in infertility: practical applications and theoretical directions for research. Eur J Obstet Gynecol Reprod Biol 2009;146:30–6. Cai J, Lou HY, Dong MY, Lu XE, Zhu YM, Gao HJ, et al. Poor ovarian response to gonadotropin stimulation is associated with low expression of follicle-stimulating hormone receptor in granulosa cells. Fertil Steril 2007;87:1350–6. The International HapMap Project. Available at: http://hapmap.ncbi.nlm.nih.gov. Accessed April 1, 2010. Knight PG, Glister C. TGF-beta superfamily members and ovarian follicle development. Reproduction 2006;132:191–206.
Vol. 94, No. 6, November 2010
Ovarian reserve, the term used to describe the capacity of the ovary to provide oocytes, reflects the quantity and quality of oocytes within the ovary at any given time (1). Diminished ovarian reserve (DOR), a decreased number of oocytes and a decrease in oocyte quality in women of reproductive age, is commonly associated with a poor response to ovarian stimulation during an in vitro fertilization (IVF) cycle (2, 3). The presence of DOR may also portend the occurrence of premature ovarian aging and Ting-Ting Wang, M.D.a,b Yan-Ting Wu, Ph.D.a,b Min-Yue Dong, Ph.D.a,b Jian-Zhong Sheng, Ph.D.c Peter C.K. Leung, Ph.D.d He-Feng Huang, M.D.a,b a Department of Reproductive Endocrinology, Zhejiang Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China b Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Hangzhou, People’s Republic of China c Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China d Department of Obstetrics and Gynecology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Received November 5, 2009; revised March 26, 2010; accepted March 29, 2010; published online May 7, 2010. T-T.W. has nothing to disclose. Y-T.W. has nothing to disclose. M-Y.D. has nothing to disclose. J-Z.S. has nothing to disclose. P.C.K.L. has nothing to disclose. H-F.H. has nothing to disclose. Supported by the China-Canada Joint Health Research Initiative Grants Program (30711120577), the National Natural Science Foundation of China (30900517), and an Innovation Award for Graduate Students of Zhejiang Province (YK2008008). Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Zhejiang Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, People’s Republic of China (FAX: 86-571-8706-1878; E-mail: [email protected]).
2490
menopause (4). Factors such as GDF-9, BMP-15, AMH, GPR3, and NOBOX are known to influence the growth and depletion rate of follicles (5, 6). The genetic variants of these factors are associated with abnormal follicular loss and might result in premature ovarian failure (POF) (6). Growth differential factor-9 (GDF-9), a member of the transforming growth factor-b superfamily, is an oocyte-derived factor that is preferentially expressed in the oocytes of humans and mice. Because GDF-9 plays a pivotal role during early folliculogenesis, deletion of GDF-9 in the mouse causes follicular arrest at the primary stage and infertility (7). Also, GDF-9 stimulates granulosa cell proliferation (8) and cumulus cell expansion (9), inhibits follicular apoptosis (10), and enhances oocyte and embryo development (11, 12). Recently, several genetic variants of GDF-9 have been identified, and their correlation with POF has been noted, suggesting that these variants contribute to aberrant follicular development and oocyte loss (13–15). We investigated the possible correlation of GDF-9 polymorphisms with DOR and evaluated the contribution of GDF-9 polymorphisms to poor IVF outcomes in women with DOR. A total of 103 women with DOR were recruited consecutively during their IVF treatment in the Reproductive Center of Zhejiang Women’s Hospital, Zhejiang University. The diagnosis of DOR was based on a (day-3) basal antral follicle count <5 combined a with basal follicle-stimulating hormone (FSH) level >10 IU/L and/or basal estradiol (E2) >80 pg/mL, as described previously elsewhere (16). All DOR diagnoses were made after at least two cycles. Other factors affecting ovarian functions were excluded: ovarian surgery, polycystic ovarian syndrome or polycystic ovaries, endometriosis, or body mass index >25. None of the women had taken hormone treatment or had endocrine disorders. The control group consisted of 123 age-matched women with normal ovarian reserve and infertility due to tubular or unexplained causes. All participants were Han Chinese from the southeast of China and had no genetic relationships. Informed consent was given by all participants for the use of their blood samples, and the ethics committee of Zhejiang University reviewed and approved this study.
Fertility and Sterility Vol. 94, No. 6, November 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.03.070
Genomic DNA was extracted from leukocytes in the peripheral blood of the women using the QiaAmp DNA Blood kit (Qiagen, Hilden, Germany). The coding regions of GDF-9 were amplified using a polymerase chain reaction (PCR) protocol (detailed information concerning the conditions is available upon request). The PCR products were sequenced using the Big Dye terminator sequencing protocol with a Big-Dye Terminator kit (Applied Biosystems, Foster City, CA) and were analyzed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Hardy-Weinberg testing for each polymorphism used Haploview version 3.2 (18). Comparisons of the distributions of genotype frequencies were performed using Fisher’s exact test or the chi-square test. Differences in clinical parameters among genotypes were tested using Student’s t-test or one-way analysis of variance for continuous variables. Categorical parameters were analyzed using Fisher’s exact test. The statistical analyses were performed with Statistical Package for the Social Sciences software version 15.0 (SPSS, Inc., Chicago, IL). P<.05 was considered statistically significant. Three variants of the GDF-9 gene, G169A, C447T (rs254286), and G546A (rs10491279), were identified by DNA sequencing. Of these, C447T and G546A are in the single nucleotide polymorphism (SNP) database, and G169A is not yet included. All these polymorphisms conformed to the Hardy-Weinberg equilibrium. A statistically significant correlation was found between the GDF-9 G546A genotype and ovarian reserve status. Thirty-four of 106 women with DOR (32.1%; CI, 23.3–41.9%) had the GA/ AA genotype compared with 24 of 123 women with normal ovarian reserve (19.5%; CI, 12.9–27.6%; P<.05).
Wang. Correspondence. Fertil Steril 2010.
Note: Values in parentheses are 95% confidence interval. MII ¼ metaphase II; rFSH ¼ recombinant follicle-stimulating hormone. a Values are mean standard deviation and compared with Student’s t-test. b Values are compared with Fisher’s exact test.
< .001 < .001 .032 < .001 < .001 .022 3037 967.2 (2,670–3,374) 64.7 (46.5–80.3) 2.41 2.33 (1.60–3.222) 23.9 (17.5–31.3) 5.3 (0.65–17.7) 6.7 (0.77–20.7) .366 .160 .436 .064 .492 .637 2371 996.6 (1,950–27,926) 33.3 (15.6–55.4) 5.29 2.03 (4.43–6.15) 71.6 (22.1–35.3) 64.7 (56.2–72.7) 36.4 (17.2–59.3) rFSH dose used (IU)a Poor ovarian response (%)b No. of MII oocytesa Fertilization rate (%)b Good quality embryos (%)b Pregnancy rate (%)b
2364 912.7 (2,182–2,546) 18.2 (11.4–27.3) 5.77 1.93 (5.39–6.16) 77.8 (75.1–80.4) 67.9 (64.5–71.2) 43 (32.8–53.8)
2,700 833.3 (2,494–2,906) 23.6 (14.4–35.5) 4.81 2.79 (4.15–5.47) 73.4 (69.4–77.2) 62.2 (57.1–67.1) 33.3 (16.4–35.3)
P value GA/AA P value GA/AA IVF outcome
GG
GG
Diminished ovarian reserve (n [ 106) Normal ovarian reserve (n [ 123)
Correlation between GDF9 G546A genotypes and in vitro fertilization outcomes in the diminished ovarian reserve patients and controls.
TABLE 1
Fertility and Sterility
The standard long down-regulation protocol of controlled ovarian stimulation (COH) was performed in all subjects as described before (17). Poor ovarian response was defined as the presence of %3 follicles with diameters of R14 mm on the day of oocyte retrieval or cycle cancellation. The presence of two pronuclei was considered to indicate fertilization. Embryos with R6 blastomeres and <20% fragmentation were considered quality embryos. Embryo transfer was performed on day 3, and a maximum of two embryos were placed in each woman. A clinical pregnancy was defined as the presence of fetal cardiac activity beyond 8 weeks of gestation.
Furthermore, we examined the correlations between polymorphisms and IVF outcomes in women with DOR or normal ovarian reserve (Table 1). In women with DOR, the carriers of the GDF-9 546A allele (GA/AA genotype) had a higher prevalence of poor ovarian response. Although these women were given more than the routine dose of recombinant FSH in controlled ovarian stimulation, they still had fewer metaphase II oocytes, a lower fertilization rate, fewer good quality embryos, and a lower pregnancy rate. In contrast, in controls, these parameters in women with the GA/ AA genotype did not differ from those with the GG genotype. However, no correlation between GDF-9 G169A or C447T and IVF outcomes was detected (data not shown). Although animal models and in vitro studies have revealed the role of GDF-9 in regulating follicular development (19), little is known about its role in human ovarian function. Our study demonstrated a strong correlation between GDF-9 G546A polymorphism in women with DOR and poor IVF outcomes, indicating that GDF9 plays an important role in determining ovarian reserve status and functions. Our findings also raise the intriguing possibility that this
2491
genetic variation of GDF-9 is responsible for the high frequency of suboptimal ovarian stimulation outcomes during IVF cycles in women with DOR. More importantly, we noted that, even though the women with the GA/AA genotype and DOR were treated with much higher doses of recombinant FSH, they still presented few good quality embryos and had a low pregnancy rate (5.3% and 6.7%), suggesting that increasing the dose of recombinant FSH is not effective in improving ovarian response and IVF outcomes in women with DOR who carry the GDF-9 546A allele. We did not detect other the GDF-9 variants such as A199C (K67E), G646A (V216M), C307T (P103S), or C557A (S186Y) that had been found in women with POF (13–15). This discrepancy may be due to our focus on patients with DOR rather than with POF. The mechanisms underlying the correlation between GDF-9 G546A polymorphism and DOR are
still unclear. Because this polymorphism is silent, there is a possibility that it has strong linkage disequilibrium with some causative sequence variants that may alter the function of GDF-9 by influencing its messenger RNA (mRNA) splicing or protein processing. Our study is the first demonstration of the role of GDF-9 genetic variants in ovarian reserve and IVF outcomes. Women with DOR and the GDF-9 G546A polymorphism showed poor IVF outcomes. These results show that the GDF-9 gene variant(s), at least in part, may be related to the poor IVF outcomes in women with DOR. Further studies are needed to clarify the mechanisms involved in the effects of these polymorphisms on ovarian functions. Acknowledgments: The authors thank Dr. Iain Bruce for reviewing and editing the manuscript, and the members of the Department of Reproductive Endocrinology in the Women’s Hospital, Zhejiang University, for their constant assistance throughout the study.
REFERENCES 1. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12:685–718. 2. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT Jr. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001;76:666–9. 3. te Velde ER, Pearson PL. The variability of female reproductive ageing. Hum Reprod Update 2002;8: 141–54. 4. Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003;18:527–33. 5. Simoni M, Tempfer CB, Destenaves B, Fauser BC. Functional genetic polymorphisms and female reproductive disorders: Part I: Polycystic ovary syndrome and ovarian response. Hum Reprod Update 2008;14:459–84. 6. Broekmans FJ, Soules MR, Fauser BC. Ovarian ageing: mechanisms and clinical consequences. Endocr Rev 2009;30:465–93. 7. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor9 is required during early ovarian folliculogenesis. Nature 1996;383:531–5.
2492
Wang et al.
Correspondence
8. Vitt UA, Hayashi M, Klein C, Hsueh AJ. Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles. Biol Reprod 2000;62: 370–7. 9. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, et al. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 2001;15:854–66. 10. Orisaka M, Orisaka S, Jiang JY, Craig J, Wang Y, Kotsuji F, et al. Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage. Mol Endocrinol 2006;20:2456–68. 11. Hussein TS, Thompson JG, Gilchrist RB. Oocytesecreted factors enhance oocyte developmental competence. Dev Biol 2006;296:514–21. 12. Yeo CX, Gilchrist RB, Thompson JG, Lane M. Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice. Hum Reprod 2008;23:67–73. 13. Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, et al. Mutations and
14.
15.
16.
17.
18.
19.
sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Eur J Endocrinol 2006;154:739–44. Chand AL, Ponnampalam AP, Harris SE, Winship IM, Shelling AN. Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure. Fertil Steril 2006;86: 1009–12. Kovanci E, Rohozinski J, Simpson JL, Heard MJ, Bishop CE, Carson SA. Growth differentiating factor-9 mutations may be associated with premature ovarian failure. Fertil Steril 2007;87:143–6. Sills ES, Alper MM, Walsh AP. Ovarian reserve screening in infertility: practical applications and theoretical directions for research. Eur J Obstet Gynecol Reprod Biol 2009;146:30–6. Cai J, Lou HY, Dong MY, Lu XE, Zhu YM, Gao HJ, et al. Poor ovarian response to gonadotropin stimulation is associated with low expression of follicle-stimulating hormone receptor in granulosa cells. Fertil Steril 2007;87:1350–6. The International HapMap Project. Available at: http://hapmap.ncbi.nlm.nih.gov. Accessed April 1, 2010. Knight PG, Glister C. TGF-beta superfamily members and ovarian follicle development. Reproduction 2006;132:191–206.
Vol. 94, No. 6, November 2010