Evaluation of the expression profile of mRNAs and lncRNAs in cumulus cells associated with polycystic ovary syndrome and pregnancy

Document Type : Original Article


1 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran

2 Reproductive Sciences and Sexual Health Research Center, Isfahan University of Medical Science, Isfahan, Iran

3 Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran

4 Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, Isfahan, Iran


Objective(s): Polycystic ovary syndrome (PCOS), the primary cause of anovulatory infertility in women, may change the gene expression profile of cumulus cells. In human ART (assisted reproductive technology), gene expression profiling in cumulus cells, a non-invasive method, may be used to identify the most competent oocytes. We aim to identify key genes according to the network-based data and assess the suitability of these genes as markers to predict oocyte competence and PCOS diagnosis.
Materials and Methods: The GSE34526 microarray dataset was obtained from the Gene Expression Omnibus (GEO) database. The function and pathway enrichment analysis for DEGs were analyzed. A protein-protein interaction (PPI) network analysis and candidate gene screening were conducted. A two-layer network consisting of mRNA and lncRNA was constructed. Expression levels of hub genes were verified using quantitative RT-PCR (qRT-PCR).
Results: A total of 2721 DEGs were retained. The PPI network of selected genes associated with the biological process of “cell communication” was analyzed, and the first 10 key genes were determined by degree. Additionally, 2 hub genes and 2 hub lncRNAs, including STAT3, RHOA, GAS5, and LINC01116, were selected from the lncRNA-mRNA network. Finally, expression levels of STAT3, RHOA, GAS5, and LINC01116 were significantly increased in the cumulus cells of PCOS patients compared to the control group (P<0.05). However, there was no significant difference in expression between the pregnant and non-pregnant groups.
Conclusion: STAT3, RHOA, GAS5, and LINC01116 may serve as possible diagnostic markers for PCOS. However, further studies on a larger population are needed to validate this finding.


Main Subjects

1. Vander Borght M, Wyns C. Fertility and infertility: Definition and epidemiology. Clinical Biochemistry 2018; 62: 2-10. 
2. Abedini M, Ghaheri A, Omani Samani R. Assisted reproductive technology in iran: The first national report on centers, 2011. Int J Fertil Steril 2016; 10: 283-289. 
3. Hoshino Y. Updating the markers for oocyte quality evaluation: Intracellular temperature as a new index. Reprod Med Biol 2018; 17: 434-441. 
4. Assidi M, Sirard MA. Cumulus cell gene expression as a marker of oocyte quality. Oogenesis 2013: 231-252. 
5. Assidi M, Montag M, Van Der Ven K, Sirard MA. Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: A preliminary study. J Assist Reprod Genet 2011; 28: 173-188. 
6. Hazekamp J, Bergh C, Wennerholm UB, Hovatta O, Karlström PO, Selbing A. Avoiding multiple pregnancies in ARTConsideration of new strategies. Hum Reprod 2000; 15: 1217-1219. 
7. Schieve LA. Multiple-gestation pregnancies after assisted reproductive technology treatment: Population trends and future directions. Women’s Heal 2007; 3: 301-307. 
8. Lee AM, Connell MT, Csokmay JM, Styer AK. Elective single embryo transfer- the power of one. Contracept Reprod Med 2016; 1: 1–7. 
9. Bromer JG, Seli E. Assessment of embryo viability in assisted reproductive technology: Shortcomings of current approaches and the emerging role of metabolomics. Curr Opin Obstet Gynecol 2008; 20: 234-241. 
10. Zhou CJ, Wu SN, Shen JP, Wang DH, Kong XW, Lu A, et al. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice. PeerJ 2016; 4: 1761-1776. 
11. Huang Z, Wells D. The human oocyte and cumulus cells relationship: New insights from the cumulus cell transcriptome. Mol Hum Reprod 2010; 16: 715-725. 
12. Sirait B, Wiweko B, Jusuf AA, Iftitah D, Muharam R. Oocyte competence biomarkers associated with oocyte maturation: A review. Front Cell Dev Biol 2021; 9: 710292-710300. 
13. Turathum B, Gao EM, Chian RC. The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization. Cells 2021; 10: 2292-2310. 
14. Khan MJ, Ullah A, Basit S. Genetic basis of polycystic ovary syndrome (PCOS): Current perspectives. Application of Clinical Genetics 2019; 12: 249-260. 
15. Ajmal N, Khan SZ, Shaikh R. Polycystic ovary syndrome (PCOS) and genetic predisposition: A review article. Eur J Obstet Gynecol Reprod Biol X 2019; 3: 100060-100066. 
16. Patel S. Polycystic ovary syndrome (PCOS), an inflammatory, systemic, lifestyle endocrinopathy. Journal of Steroid Biochemistry and Molecular Biology 2018; 182: 27-36. 
17. De Leo V, Musacchio MC, Cappelli V, Massaro MG, Morgante G, Petraglia F. Genetic, hormonal and metabolic aspects of PCOS: An update. Reprod Biol Endocrinol 2016; 14: 1–17. 
18. Jansen E, Laven JSE, Dommerholt HBR, Polman J, Van Rijt C, Van Den Hurk C, et al. Abnormal gene expression profiles in human ovaries from polycystic ovary syndrome patients. Mol Endocrinol 2004; 18: 3050-3063. 
19. Li J, Cao Y, Xu X, Xiang H, Zhang Z, Chen B, et al. Increased new lncRNA-mRNA gene pair levels in human cumulus cells correlate with oocyte maturation and embryo development. Reprod Sci 2015; 22: 1008-1014. 
20. Qin L, Huang CC, Yan XM, Wang Y, Li ZY, Wei XC. Long non-coding RNA h19 is associated with polycystic ovary syndrome in Chinese women: A preliminary study. Endocr J 2019; 66: 587-595. 
21. Alawieh A, Zaraket FA, Li JL, Mondello S, Nokkari A, Razafsha M, et al. Systems biology, bioinformatics, and biomarkers in neuropsychiatry. Front Neurosci 2012; 6: 187-203. 
22. Li SH, Lin MH, Hwu YM, Lu CH, Yeh LY, Chen YJ, et al. Correlation of cumulus gene expression of GJA1, PRSS35, PTX3, and SERPINE2 with oocyte maturation, fertilization, and embryo development. Reprod Biol Endocrinol 2015; 13: 1–8. 
23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402-408. 
24. Feuerstein P, Cadoret V, Dalbies-Tran R, Guerif F, Bidault R, Royere D. Gene expression in human cumulus cells: One approach to oocyte competence. Hum Reprod 2007; 22: 3069–3077. 
25. Papler TB, Bokal EV, Lovrecic L, Kopitar AN, Maver A. No specific gene expression signature in human granulosa and cumulus cells for prediction of oocyte fertilisation and embryo implantation. PLoS One 2015; 10: e0115865-e0115878. 
26. Hart R, Norman R. Polycystic ovarian syndrome--prognosis and outcomes. Best Pract Res Clin Obstet Gynaecol 2006; 20: 751–778. 
27. Shen H, Liang Z, Zheng S, Li X. Pathway and network-based analysis of genome-wide association studies and RT-PCR validation in polycystic ovary syndrome. Int J Mol Med 2017; 40: 1385–1396. 
28. Eftekhar M, Mohammadian F, Yousefnejad F, Molaei B, Aflatoonian A. Comparison of conventional IVF versus ICSI in non-male factor, normoresponder patients. Iran J Reprod Med 2012; 10: 131-136. 
29. Boulet SL, Mehta A, Kissin DM, Lee W, Kawwass JF, Jamieson DJ. Trends in use of and reproductive outcomes associated with intracytoplasmic sperm injection. JAMA 2015; 313: 255–263. 
30. Tannus S, Son WY, Gilman A, Younes G, Shavit T, Dahan MH. The role of intracytoplasmic sperm injection in non-male factor infertility in advanced maternal age. Hum Reprod 2017; 32: 119–124. 
31. Gennarelli G, Carosso A, Canosa S, Filippini C, Cesarano S, Scarafia C, et al. ICSI versus conventional IVF in women aged 40 years or more and unexplained infertility: A retrospective evaluation of 685 cycles with propensity score model. J Clin Med 2019; 8: 1694-1705. 
32. Wen L, Craig J, Dyce PW, Li J. Cloning of porcine signal transducer and activator of transcription 3 cDNA and its expression in reproductive tissues. Reproduction 2006; 132: 511–518. 
33. Shao S, Wang H, Shao W, Liu N. MiR-199a-5p stimulates ovarian granulosa cell apoptosis in polycystic ovary syndrome. J Mol Endocrinol 2020; 65: 187–201. 
34. Li MG, Ding GL, Chen XJ, Lu XP, Dong LJ, Dong MY, et al. Association of serum and follicular fluid leptin concentrations with granulosa cell phosphorylated signal transducer and activator of transcription 3 expression in fertile patients with polycystic ovarian syndrome. J Clin Endocrinol Metab 2007; 92: 4771–4776. 
35. Ou-Yang H, Wu SC, Sung LY, Yang SH, Yang SH, Chong KY, et al. STAT3 is an upstream regulator of granzyme G in the maternal-to-zygotic transition of mouse embryos. Int J Mol Sci 2021; 22: 1–19. 
36. Liu Z, De Matos DG, Fan HY, Shimada M, Palmer S, Richards JAS. Interleukin-6: An autocrine regulator of the mouse cumulus cell-oocyte complex expansion process. Endocrinology 2009; 150: 3360–3368. 
37. Antczak M, Van Blerkom J. Oocyte influences on early development: The regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 1997; 3: 1067–1086. 
38. Robker RL, Watson LN, Robertson SA, Dunning KR, McLaughlin EA, Russell DL. Identification of sites of STAT3 action in the female reproductive tract through conditional gene deletion. PLoS One 2014; 9: e101182-101198. 
39. El Zowalaty AE, Li R, Zheng Y, Lydon JP, DeMayo FJ, Ye X. Deletion of RhoA in progesterone receptor–expressing cells leads to luteal insufficiency and infertility in female mice. Endocrinology 2017; 158: 2168-2178. 
40. Liu L, Wang Y, Yu Q. The PI3K/Akt signaling pathway exerts effects on the implantation of mouse embryos by regulating the expression of RhoA. Int J Mol Med 2014; 33: 1089-1096. 
41. Liu X, Yan F, Yao H, Chang M, Qin J, Li Y, et al. Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture. Cell Tissue Res 2014; 358: 359–369. 
42. Zhong ZS, Huo LJ, Liang CG, Chen DY, Sun QY. Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage. Mol Reprod Dev 2005; 71: 256–261. 
43. Budna J, Bryja A, Celichowski P, Kahan R, Kranc W, Ciesiółka S, et al. Genes of cellular components of morphogenesis in porcine oocytes before and after IVM. Reproduction 2017; 154: 535–545. 
44. Lin H, Xing W, Li Y, Xie Y, Tang X, Zhang Q. Downregulation of serum long noncoding RNA GAS5 may contribute to insulin resistance in PCOS patients. Gynecol Endocrinol 2018; 34: 784–788. 
45. Mu L, Sun X, Tu M, Zhang D. Non-coding RNAs in polycystic ovary syndrome: A systematic review and meta-analysis. Reproductive Biology and Endocrinology 2021; 19: 1–18. 
46. Wang C, Yue S, Jiang Y, Mao Y, Zhao Z, Liu X, et al. LncRNA GAS5 is upregulated in polycystic ovary syndrome and regulates cell apoptosis and the expression of IL-6. J Ovarian Res 2020; 13: 1-8. 
47. Wang J, Gong X, Tian GG, Hou C, Zhu X, Pei X, et al. Long noncoding RNA growth arrest-specific 5 promotes proliferation and survival of female germline stem cells in vitro. Gene 2018; 653: 14–21. 
48. Battaglia R, Vento ME, Borzì P, Ragusa M, Barbagallo D, Arena D, et al. Non-coding RNAs in the ovarian follicle. Front Genet 2017; 8: 57-68. 
49. Zeng L, Lyu X, Yuan J, Wang W, Zhao N, Liu B, et al. Long non-coding RNA LINC01116 is overexpressed in lung adenocarcinoma and promotes tumor proliferation and metastasis. Am J Transl Res 2020; 12: 4302-4313. 
50. Xu Y, Yu X, Zhang M, Zheng Q, Sun Z, He Y, et al. Promising advances in LINC01116 related to cancer. Front Cell Dev Biol 2021; 9: 736927-736937.