Altered Expression of GDF9 and BMP15 Genes in Granulosa Cells of Diminished Ovarian Reserve Patients: A Case-Control Study

Document Type : Original Article


1 Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

3 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran,Iran

4 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

5 Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

6 Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

7 Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada

8 Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, London, Ontario, Canada


Objective: Diminished ovarian reserve (DOR) is a challenging issue encountered during assisted reproductive technology. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) belong to the transforming growth factor-beta (TGF-β) superfamily which are essential for folliculogenesis. We aimed to the evaluation
of the GDF9 and BMP15 expression in the granulosa cells (GCs) of DOR patients.
Materials and Methods: This case-control study included 14 women with DOR and 12 controls, who were between 28-40 years of age undergoing controlled ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol. DOR patients were selected by the Bologna criteria. The GCs were extracted from the aspirated follicular fluids and RNA isolated from this. The fold change of gene expressions was assessed by real-time polymerase chain reaction (PCR).
Results: GDF9 expression in patients was 0.23 times lower than the control group, which was significant (P<0.0001). BMP15 expression in patients was 0.32 times lower than the control group, which was significant (P<0.0001). The number of archived oocytes, MII, and two pronuclei (PN) embryos was higher in the control group and these differences were statistically significant (P<0.05).
Conclusion: Given that GDF9 and BMP15 are specifically involved during follicular recruitmen., we expect expression of these two genes in DOR patients which is greatly reduced by reducing follicular reserve.


1. May-Panloup P, Ferré-L’Hôtellier V, Morinière C, Marcaillou C, Lemerle S, Malinge MC, et al. Molecular characterization of corona radiata cells from patients with diminished ovarian reserve using microarray and microfluidic-based gene expression profiling. Hum Reprod. 2012; 27(3): 829-843.
2. Greene AD, Patounakis G, Segars JH. Genetic associations with diminished ovarian reserve: a systematic review of the literature. J Assist Reprod Genet. 2014; 31(8): 935-946.
3. Fusco F, Paciolla M, Chen E, Li X, Genesio R, Conti A, et al. Genetic and molecular analysis of a new unbalanced X; 18 rearrangement: localization of the diminished ovarian reserve disease locus in the distal Xq POF1 region. Hum Reprod. 2011; 26(11): 3186-3196.
4. Jahromi BN, Sadeghi S, Alipour S, Parsanezhad ME, Alamdarloo SM. Effect of melatonin on the outcome of assisted reproductive technique cycles in women with diminished ovarian reserve: a double-blinded randomized clinical trial. Iran J Med Sci. 2017; 42(1): 73-78.
5. Sun W, Stegmann BJ, Henne M, Catherino WH, Segars JH. A new approach to ovarian reserve testing. Fertil Steril. 2008; 90(6): 2196-2202.
6. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril. 2001; 76(4): 666-669.
7. Ferraretti A, La Marca A, Fauser B, Tarlatzis B, Nargund G, Gianaroli L, et al. ESHRE consensus on the definition of ‘poor
response’to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011; 26(7): 1616-1624.
8. de Castro FC, Cruz MHC, Leal CLV. Role of growth differentiation factor 9 and bone morphogenetic protein 15 in ovarian function and their importance in mammalian female fertility—a review. Asian-Australas J Anim Sci. 2016; 29(8): 1065-1074.
9. Thomas FH, Vanderhyden BC. Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth. Reprod Biol Endocrinol. 2006; 4(1): 19.
10. Ceko M, Hummitzsch K, Hatzirodos N, Bonner W, Aitken J, Russell D, et al. X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function. Metallomics. 2014; 7(1): 71-82.
11. 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(4): 1009-1012.
12. Juengel J, McNatty K. The role of proteins of the transforming growth factor-β superfamily in the intraovarian regulation of follicular development. Hum Reprod Update. 2005; 11(2): 144-161.
13. Silva J, Van den Hurk R, Van Tol H, Roelen B, Figueiredo J. Expression of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors in the ovaries of goats. Mol Reprod Dev. 2005; 70(1): 11-19.
14. Su YQ, Sugiura K, Wigglesworth K, O’Brien MJ, Affourtit JP, Pangas SA, et al. Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells. Development. 2008; 135(1): 111-121.
15. Wang TT, Ke ZH, Song Y, Chen LT, Chen XJ, Feng C, et al. Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women. Hum Reprod. 2013; 28(9): 2473-2481.
16. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature. 1996; 383(6600): 531-535.
17. Otsuka F, McTavish KJ, Shimasaki S. Integral role of GDF-9 and BMP-15 in ovarian function. Mol Reprod Dev. 2011; 78(1): 9-21.
18. McMahon HE, Hashimoto O, Mellon PL, Shimasaki S. Oocytespecific overexpression of mouse bone morphogenetic protein-15 leads to accelerated folliculogenesis and an early onset of acyclicity in transgenic mice. Endocrinology. 2008; 149(6): 2807-2815.
19. McNatty KP, Moore L, Hudson NL, Quirke L, Lawrence SB, Reader K, et al. The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology. Reproduction. 2004; 128(4): 379-386.
20. Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocr Rev. 2004; 25(1): 72-101.
21. Paulini F, Melo EO. The role of oocyte-secreted factors GDF9 and BMP15 in follicular development and oogenesis. Reprod Domest Anim. 2011; 46(2): 354-361.
22. Balaban B, Brison D, Calderon G, Catt J, Conaghan J, Cowan L, et al. Alpha scientists in reproductive medicine and ESHRE special interest group of embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011; 26(6): 1270-1283.
23. Greenseid K, Jindal S, Hurwitz J, Santoro N, Pal L. Differential granulosa cell gene expression in young women with diminished ovarian reserve. Reprod Sci. 2011; 18(9): 892-899.
24. Chilvers R, Bodenburg Y, Denner L, Urban R. Development of a novel protocol for isolation and purification of human granulosa cells. J Assist Reprod Genet. 2012; 29(6): 547-556.
25. Aghadavod E, Zarghami N, Farzadi L, Zare M, Barzegari A, Movassaghpour AA, et al. Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments. Adv Biomed Res. 2015; 4.
26. Thomas FH, Vanderhyden BC. Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth. Reprod Biol Endocrinology. 2006;4(1):1-8.
27. Sudiman J, Sutton-McDowall ML, Ritter LJ, White MA, Mottershead DG, Thompson JG, et al. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence. PLoS One. 2014; 9(7): e103563.
28. Sugimura S, Ritter LJ, Sutton-McDowall ML, Mottershead DG, Thompson JG, Gilchrist RB. Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply. Mol Hum Reprod. 2014; 20(6): 499-513.
29. Wang TT, Wu YT, Dong MY, Sheng JZ, Leung PC, Huang HF. G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve. Fertil Steril. 2010; 94(6): 2490-2492.
30. Gong Y, Li-Ling J, Xiong D, Wei J, Zhong T, Tan H. Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders. J Ovarian Res. 2021; 14(1): 1-10.