CALM2 gene expression levels in the cumulus cells as a marker for chromosomal abnormalities in the embryo in in vitro fertilization programs

Safronova N.A., Kalinina E.A., Donnikov A.E., Burmenskaya O.V., Makarova N.P., Gorshinova V.K.

Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia, Moscow 117997, Ac. Oparina str. 4, Russia
Objective. To search for molecular genetic markers associated with chromosomal abnormalities in embryos in order to optimize the selection of transferred embryos to enhance the efficiency of infertility treatment using assisted reproductive technologies.
Subjects and methods. 42 cumulus cell samples from 11 patients who had undergone an in vitro fertilization (IVF) (intracytoplasmic sperm injection, ICSI) + preimplantation genetic screening (PGS) program were analyzed during the conducted study. The obtained embryos were divided into 4 groups according to the presence of their chromosomal abnormalities: 1) embryos without chromosomal abnormalities (20 samples); 2) those with sex chromosomal aneuploidies (n = 3); 3) those with somatic chromosomal aneuploidies (n =15); 4) heteroploid embryos (n = 4). A real-time RT-PCR assay was used to examine the cumulus cell mRNA expression in 10 genes: hyaluronan synthase 2 (HAS2), prostaglandin synthase 2 (PTFS2), gremlin 1 (GREM1), versican (VCAN), inositol-triphosphate 3 kinase A (ITPKA), activated leukocyte cell adhesion molecules (ALCAM or CD 166), syndecan 4 (SDC4), calmodulin 2 (CALM2), SPRY domain-containing suppressor of cytokine signaling box protein 2 (SPSB2), and tumor protein 5313 (TP5313).
Results. There was a relationship between the mRNA expression level in the СD166 (ALCAM), SDC4, VCAN and CALM2 genes and the presence of chromosomal abnormalities of the embryo (p = 0.004, p = 0.009, p = 0.042, p = 0.054, respectively). Discriminant analysis showed that the mRNA expression level in the CALM2 gene is the most informative marker for chromosomal abnormalities in the embryos in IVF programs. The study revealed that the poor embryo quality group showed chromosomal abnormalities in 78.6% of cases (OR = 5.7 (1.4 to 23.9); p = 0.023). There were no statistically significant differences between the mRNA expression level in the studied genes and the quality of developing embryos (p> 0.05).
Conclusion. The mRNA expression level in the CALM2 gene is the most informative marker for chromosomal abnormalities in the embryos in IVF programs. However, there is a need for further investigation of the gene expression profile to search for potential biomarkers in the cumulus cells in order to create a prognostic model that can highly accurately predict the probability of chromosomal abnormalities in the embryos. This mathematical model will be able to further optimize the selection of transferred embryos, thereby enhancing the effectiveness of IVF programs as a whole.

Keywords

infertility
cumulus cells
gene expressions
aneuploidies
in vitro fertilization

References

1. de Mouzon J., Goossens V., Bhattacharya S., Castilla J.A., Ferraretti A.P., Korsak V. et al. Assisted reproductive technology in Europe, 2007: results generated from European registers by ESHRE. Hum. Reprod. 2012; 27(4): 954-66.

2. Smolnikova V.Yu., Kalinina E.A., Krasnoshchoka O.E., Donnikov A.E., Burmenskaya O.E., Trofimov D.Yu., Sukhikh G.T. Possibilities for noninvasive oocyte and embryo evaluation when implementing assisted reproductive technology programs for follicular-fluid growth factor mRNA expression. Akusherstvo i Ginekologiya/Obstetrics and Gynecology. 2014; (9): 36-43. (in Russian)

3. Poli M., Ori A., Child T., Jaroudi S., Spath K., Beck M., Wells D. Characterization and quantification of proteins secreted by single human embryos prior to implantation. EMBO Mol. Med. 2015; 7(11): 1465-79.

4. Ebner T., Moser M., Sommergruber M., Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum. Reprod. Update. 2003; 9(3): 251-62.

5. Yang Z., Liu J., Collins G.S., Salem S.A., Liu X., Lyle S.S. et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol. Cytogenet. 2012; 5(1): 24.

6. Gromenko Yu.Yu., Ishakov I.R. The impact of quality assessment factors in the prediction of the transferred embryo pregnancy rate in in vitro fertilization programs. Meditsinskiy vestnik Bashkortostana. 2012; 7(2): 27-30. (in Russian)

7. Harton G., Braude P., Lashwood A., Schmutzler A., Traeger-Synodinos J., Wilton L., Harper J.C. ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening. Hum. Reprod. 2011; 26(1): 14-24.

8. Scott R.T. Jr., Upham K.M., Forman E.J., Hong K.H., Scott K.L., Taylor D.T. et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil. Steril. 2013; 100(3): 697-703.

9. Cohen J., Wells D., Munné S. Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertil. Steril. 2007; 87(3): 496-503.

10. Assou S., Haouzi D., De Vos J., Hamamah S. Human cumulus cells as biomarkers for embryo and pregnancy outcomes. Mol. Hum. Reprod. 2010; 16(8): 531-8.

11. Kõks S., Velthut A., Sarapik A., Altmäe S., Reinmaa E., Schalkwyk L.C. et al. The differential transcriptome and ontology profiles of floating and cumulus granulosa cells in stimulated human antral follicles. Mol. Hum. Reprod. 2010; 16(4): 229-40.

12. Blashkiv T.B., Shepel A.A., Voznesenskaya T.Yu. E Gene expression of cumulus cells surrounding the oocyte during ovulation and fertilization (review). Problemyi reproduktsii. 2014; 20(1): 55-8. (in Russian)

13. Adriaenssens T., Segers I., Wathlet S., Smitz J. The cumulus cell gene expression profile of oocytes with different nuclear maturity and potential for blastocyst formation. J. Assist. Reprod. Genet. 2011; 28(1): 31-40.

14. Anderson R.A., Sciorio R., Kinnell H., Bayne R.A., Thong K.J., de Sousa P.A., Pickering S. Cumulus gene expression as a predictor of human oocyte fertilisation, embryo development and competence to establish a pregnancy. Reproduction. 2009; 138(4): 629-37.

15. McKenzie L.J., Pangas S.A., Carson S.A., Kovanci E., Cisneros P., Buster J.E. et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum. Reprod. 2004; 19(12): 2869-74.

16. Gilchrist R.B., Lane M., Thompson J.G. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum. Reprod. Update. 2008; 14(2): 159-77.

17. Tanghe S., Van Soom A., Nauwynck H., Coryn M., de Kruif A. Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol. Reprod. Dev. 2002; 61(3): 414-24.

18. Binelli M., Murphy B.D. Coordinated regulation of follicle development by germ and somatic cells. Reprod. Fertil. Dev. 2010; 22(1): 1-12.

19. Thomas F.H., Ethier J.F., Shimasaki S., Vanderhyden B.C. Follicle-stimulating hormone regulates oocyte growth by modulation of expression of oocyte and granulosa cell factors. Endocrinology. 2005; 146(2): 941-9.

20. Burnik Papler T., Vrtacnik Bokal E., Maver A., Kopitar A.N., Lovrečić L. Transcriptomic analysis and meta-analysis of human granulosa and cumulus cells. PLoS One. 2015; 10(8): e0136473.

21. Wathlet S., Adriaenssens T., Segers I., Verheyen G., Van de Velde H., Coucke W. et al. Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Hum. Reprod. 2011; 26(5): 1035-51.

22. Gebhardt K.M., Feil D.K., Dunning K.R., Lane M., Russell D.L. Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer. Fertil. Steril. 2011; 96(1): 47-52. e2.

23. Wathlet S., Adriaenssens T., Segers I., Verheyen G., Janssens R., Coucke W. et al. New candidate genes to predict pregnancy outcome in single embryo transfer cycles when using cumulus cell gene expression. Fertil. Steril. 2012; 98(2): 432-9. e1-4.

24. Fragouli E., Wells D., Iager A.E., Kayisli U.A., Patrizio P. Alteration of gene expression in human cumulus cells as a potential indicator of oocyte aneuploidy. Hum. Reprod. 2012; 27(8): 2559-68.

25. 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): 1270-83.

26. Marei W.F.A., Salavati M., Fouladi-Nashta A.A. Critical role of hyaluronidase-2 during preimplantation embryo development. Mol. Hum. Reprod. 2013; 19(9): 590-9.

27. Shimada M., Yanai Y., Okazaki T., Noma N., Kawashima I., Mori T., Richards J.S. Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization. Development. 2008; 135(11): 2001-11.

28. Alaniz L., Rizzo M., Garcia M.G., Piccioni F., Aquino J.B., Malvicini M. et al. Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma. Cancer Immunol. Immunother. 2011; 60(10): 1383-95.

29. Sathyan S., Koshy L.V., Balan S., Easwer H.V., Premkumar S., Nair S. et al. Association of Versican (VCAN) gene polymorphisms rs251124 and rs2287926 (G428D), with intracranial aneurysm. Meta Gene. 2014; 2: 651-60.

30. Theocharis A.D. Versican in health and disease. Connect. Tissue Res. 2008; 49(3): 230-4.

31. Arosh J.A., Banu S.K., Chapdelaine P., Fortier M.A. Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy.Endocrinology. 2004; 145(1): 407-17.

32. Pangas S.A., Jorgez C.J., Matzuk M.M. Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin. J. Biol. Chem. 2004; 279(31): 32281-6.

33. Sutton A., Friand V., Brulé-Donneger S., Chaigneau T., Ziol M., Sainte-Catherine O. et al. Stromal cell-derived factor-1/chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion. Mol. Cancer Res. 2007; 5(1): 21-33.

34. Kojima T., Katsumi A., Yamazaki T., Muramatsu T., Nagasaka T., Ohsumi K., Saito H. Human ryudocan from endothelium-like cells binds basic fibroblast growth factor, midkine, and tissue factor pathway inhibitor. J. Biol. Chem. 1996; 271(10): 5914-20.

35. Sakata M., Kobayashi H., Sun G.W., Mochizuki O., Takagi A., Kojima T. Ryudocan expression by luteinized granulosa cells is associated with the process of follicle atresia. Fertil. Steril. 2000; 74(6): 1208-14.

36. Prevarskaya N., Skryma R., Shuba Y. Calcium in tumour metastasis: new roles for known actors. Nat. Rev. Cancer. 2011; 11(8): 609-18.

37. Katz A.M. Cardiac ion channels. N. Engl. J. Med. 1993; 328: 1244-51.

38. Berridge M.J., Lipp P., Bootman M.D. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 2000; 1(1): 11-21.

39. Yakovets O.G. Stress phytophysiology. Lecture course. Minsk: BSU; 2010. 103c. (in Russian)

40. Severin S.E., ed. Biological chemistry with exercises and tasks. Moscow: GEOTAR-Media; 2011. 624p. (in Russian)

41. Kulasingam V., Zheng Y., Soosaipillai A., Leon A.E., Gion M., Diamandis E.P. Activated leukocyte cell adhesion molecule: a novel biomarker for breast cancer. Int. J. Cancer. 2009; 125(1): 9-14.

42. Hernandez-Gonzalez I., Gonzalez-Robayna I., Shimada M., Wayne C.M., Ochsner S.A., White L., Richards J.S. Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? Mol. Endocrinol. 2006; 20(6): 1300-21.

43. Yilmaz G., Granger D.N. Cell adhesion molecules and ischemic stroke. Neurol. Res. 2008; 30(8): 783-93.

44. Matsumoto H., Ma W.G., Daikoku T., Zhao X., Paria B.C., Das S.K. et al. Cyclooxygenase-2 differentially directs uterine angiogenesis during implantation in mice. J. Biol. Chem. 2002; 277(32): 29260-7.

45. Porté S., Valencia E., Yakovtseva E.A., Borràs E., Shafqat N., Debreczeny J.E. et al. Three-dimensional structure and enzymatic function of proapoptotic human p53-inducible quinone oxidoreductase PIG3. J. Biol. Chem. 2009; 284(25): 17194-205.

46. Lee J.H., Kang Y., Khare V., Jin Z.Y., Kang M.Y., Yoon Y. et al. The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage. Oncogene. 2010; 29(10): 1431-50.

47. Kuang Z., Lewis R.S., Curtis J.M., Zhan Y., Saunders B.M., Babon J.J. et al. The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation. J. Cell. Biol. 2010: 190(1): 129-41.

Received 20.05.2016

Accepted 27.05.2016

About the Authors

Safronova Natalia Aleksandrovna, M.D., postgraduate of the department of assisted reproductive technology in infertility treatment, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954382501. E-mail: safronchik9090@bk.ru
Kalinina Elena Anatolievna, M.D, Ph.D, The Head of the department of assisted reproductive technology in infertility treatment, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954381341. E-mail: e_kalinina@oparina4.ru
Donnikov Andrew Evgenievich, PhD, Senior Researcher of molecular-genetical laboratory, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954381341. E-mail: a_donnikov@oparina4.ru
Bourmenskaya Olga Vladimirovna, Ph.D., Researcher of molecular-genetical laboratory, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954381341. E-mail: o_bourmenskaya@oparina4.ru
Makarova Natalia Petrovna, PhD, Researcher of the department of assisted reproductive technology in infertility treatment, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954381341. E-mail: np.makarova@gmail.com
Gorshinova Viсtoria Konstantinovna, M.D., postgraduate of the department of assisted reproductive technology in infertility treatment, Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of Russia. 117997, Russia, Moscow, Ac. Oparina 4. Tel.: +74954382501. E-mail: chiasma@mail.ru

For citations: Safronova N.A., Kalinina E.A., Donnikov A.E., Burmenskaya O.V., Makarova N.P., Gorshinova V.K. CALM2 gene expression levels in the cumulus cells as a marker for chromosomal abnormalities in the embryo in in vitro fertilization programs. Akusherstvo i Ginekologiya/Obstetrics and Gynecology. 2016; (10): 64-72. (in Russian)
http://dx.doi.org/10.18565/aig.2016.10.64-72

Similar Articles