Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Elevated Levels of Serum Vascular Endothelial Growth Factor-A Are Not Related to NK Cell Parameters in Recurrent IVF Failure

Authors
1 Department of Immunology and Allergy, St Helier Hospital, Carshalton, United Kingdom
2 Fertility Clinic, Lister Hospital, London, United Kingdom
Abstract
Background: Vascular Endothelial Growth Factor and NK cells have an inter-related role in angiogenesis that is critical for placentation and success of in vitro fertilization. An attempt was made to assess a possible relationship between the two in this study. Methods: A case control study was performed comparing the serum levels of VEGF-A and its receptor VEGF-R1 with levels of NK cells, activated NK cells and NK cytotoxicity in 62 women with Repeated Implantation Failure (RIF). The healthy control group consisted of 72 women of similar age, without known issues in achieving pregnancy or evidence of autoimmunity. Levels of VEGF-A and VEGF-R1 were quantified by ELISA methods with standard curve interpolation. NK cell subsets were determined with flow cytometry using fluorescent-tagged anti-CD56, anti-CD16, anti-CD3 and anti-CD69. NK cytotoxicity was performed by incubating peripheral blood mononuclear cells and K562 cultured cells with propidium iodide, steroid, intralipid and intravenous immunoglobulin, using previously described methods. Statistical analysis involved Mann-Whitney-U and Spearman’s rank correlation testing with p-values defined as <0.05. Results: It was found that VEGF-A levels were significantly raised in women with RIF compared to healthy controls (362.9 vs. 171.6 pg/ml, p<0.0001), with no difference in VEGF-R1 levels between groups (1499 vs. 1202 pg/ml, p=0.4082). There was no correlation between VEGF-A or VEGF-R1 and the absolute levels of circulating NK cells, CD69 activated NK cells or NK cytotoxicity. Conclusion: The absence of correlation between VEGF-A or VEGF-R1 and NK cells suggests VEGF secretion and regulation is independent of NK cell activity in RIF.
Keywords

  1. Tan BK, Vandekerckhove P, Kennedy R, Keay SD. Investigation and current management of recurrent IVF treatment failure in the UK. BJOG. 2005;112(6):773-80.
  2. Polanski LT, Barbosa MA, Martins WP, Baumgarten MN, Campbell B, Brosens J, et al. Interventions to improve reproductive outcomes in women with elevated natural killer cells undergoing assisted reproduction techniques: a systematic review of literature. Hum Reprod. 2014;29(1):65-75.
  3. Llurba E, Crispi F, Verlohren S. Update on the pathophysiological implications and clinical role of angiogenic factors in pregnancy. Fetal Diagn Ther. 2015;37(2):81-92.
  4. Muttukrishna S, Suri S, Groome N, Jauniaux E. Relationships between TGFbeta proteins and oxygen concentrations inside the first trimester human gestational sac. PLoS One. 2008;3(6):e2302.
  5. Weimar CH, Kavelaars A, Brosens JJ, Gellersen B, de Vreeden-Elbertse JM, Heijnen CJ, et al. Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high- and low-quality human embryos. PLoS One. 2012;7(7):e41424.
  6. Duffy AM, Bouchier-Hayes DJ, Harmey JH. Vascular endothelial growth factor (VEGF) and its role in non-endothelial cells: Autocrine signalling by VEGF. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6482/
  7. Li XF, Charnock-Jones DS, Zhang E, Hiby S, Malik S, Day K, et al. Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells. J Clin Endocrinol Metab. 2001;86(4):1823-34.
  8. Charnock-Jones DS, Kaufmann P, Mayhew TM. Aspects of human fetoplacental vasculogenesis and angiogenesis. I. Molecular regulation. Placenta. 2004;25(2-3):103-13.
  9. Taylor CM, Stevens H, Anthony FW, Wheeler T. Influence of hypoxia on vascular endothelial growth factor and chorionic gonadotrophin production in the trophoblast-derived cell lines: JEG, JAr and BeWo. Placenta. 1997;18(5-6):451-8.
  10. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem. 1994;269(43):26988-95.
  11. Gerber HP, Condorelli F, Park J, Ferrara N. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol Chem. 1997;272(38):23659-67.
  12. Ratsep MT, Felker AM, Kay VR, Tolusso L, Hofmann AP, Croy BA. Uterine natural killer cells: supervisors of vasculature construction in early decidua basalis. Reproduction. 2015;149(2):R91-102.
  13. Robson A, Harris LK, Innes BA, Lash GE, Aljunaidy MM, Aplin JD, et al. Uterine natural killer cells initiate spiral artery remodeling in human pregnancy. FASEB J. 2012;26(12):4876-85.
  14. Fu B, Li X, Sun R, Tong X, Ling B, Tian Z, et al. Natural killer cells promote immune tolerance by regulating inflammatory TH17 cells at the human maternal-fetal interface. Proc Natl Acad Sci USA. 2013;110(3):E231-40.
  15. Gao Y, Wang PL. Increased CD56(+) NK cells and enhanced Th1 responses in human unexplained recurrent spontaneous abortion. Genet Mol Res. 2015;14(4):18103-9.
  16. Karami N, Boroujerdnia MG, Nikbakht R, Khodadadi A. Enhancement of peripheral blood CD56(dim) cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure. J Reprod Immunol. 2012;95(1-2):87-92.
  17. Ghafourian M, Karami N, Khodadadi A, Nikbakht R. Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure. Iran J Immunol. 2014;11(2):84-96.
  18. Thum MY, Bhaskaran S, Abdalla HI, Ford B, Sumar N, Shehata H, et al. An increase in the absolute count of CD56dimCD16+CD69+ NK cells in the peripheral blood is associated with a poorer IVF treatment and pregnancy outcome. Hum Reprod. 2004;19(10):2395-400.
  19. Papamitsou T, Toskas A, Papadopoulou K, Sioga A, Lakis S, Chatzistamatiou M, et al. Immunohistochemical study of immunological markers: HLAG, CD16, CD25, CD56 and CD68 in placenta tissues in recurrent pregnancy loss. Histol Histopathol. 2014;29(8):1047-55.
  20. Wang S, Li YP, Ding B, Zhao YR, Chen ZJ, Xu CY, et al. Recurrent miscarriage is associated with a decline of decidual natural killer cells expressing killer cell immunoglobulin-like receptors specific for human leukocyte antigen C. J Obstet Gynaecol Res. 2014;40(5):1288-95.
  21. Almasry SM, Elmansy RA, Elfayomy AK, Algaidi SA. Ultrastructure alteration of decidual natural killer cells in women with unexplained recurrent miscarriage: a possible association with impaired decidual vascular remodelling. J Mol Histol. 2015;46(1):67-78.
  22. Jakovljevic A, Bogavac M, Lozanov-Crvenkovic Z, Milosević-Tosic M, Nikolic A, Mitic G. Early pregnancy angiogenic proteins levels and pregnancy related hypertensive disorders. J Matern Fetal Neonatal Med. 2017;30(5):534-9.
  23. Andraweera PH, Dekker GA, Roberts CT. The vascular endothelial growth factor family in adverse pregnancy outcomes. Hum Reprod Update. 2012;18(4):436-57.
  24. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003;111(5):649-58.
  25. Keskin U, Ulubay M, Dede M, Ozgurtas T, Koçyigit YK, Aydin FN, et al. The relationship between the VEGF/sVEGFR-1 ratio and threatened abortion. Arch Gynecol Obstet. 2015;291(3):557-61.
  26. Pang L, Wei Z, Li O, Huang R, Qin J, Chen H, et al. An increase in vascular endothelial growth factor (VEGF) and VEGF soluble receptor-1 (sFlt-1) are associated with early recurrent spontaneous abortion. PLoS One. 2013;8(9):e75759.
  27. Xu X, Du C, Li H, Du J, Yan X, Peng L, et al. Association of VEGF genetic polymorphisms with recurrent spontaneous abortion risk: a systematic review and meta-analysis. PLoS One. 2015;10(4):e0123696.
  28. Yalcintepe SA, Silan F, Hacivelioglu SO, Uludag A, Cosar E, Ozdemir O. Fetal Vegf Genotype is More Important for Abortion Risk than Mother Genotype. Int J Mol Cell Med. 2014;3(2):88-94.
  29. Chiossone L, Vacca P, Orecchia P, Croxatto D, Damonte P, Astigiano S, et al. In vivo generation of decidual natural killer cells from resident hematopoietic progenitors. Haematologica. 2014;99(3):448-57.
  30. Lima PD, Tu MM, Rahim MM, Peng AR, Croy BA, Makrigiannis AP. Ly49 receptors activate angiogenic mouse DBA⁺ uterine natural killer cells. Cell Mol Immunol. 2014;11(5):467-76.
  31. Zhang J, Dunk CE, Lye SJ. Sphingosine signalling regulates decidual NK cell angiogenic phenotype and trophoblast migration. Hum Reprod. 2013;28(11):3026-37.
  32. Chiba H, Fukui A, Fuchinoue K, Funamizu A, Tanaka K, Mizunuma H. Expression of Natural Cytotoxicity Receptors on and Intracellular Cytokine Production by NK Cells in Women with Gestational Diabetes Mellitus. Am J Reprod Immunol. 2016;75(5):529-38.
  33. Mosimann B, Wagner M, Shehata H, Poon LC, Ford B, Nicolaides KH, et al. Natural killer cells and their activation status in normal pregnancy. Int J Reprod Med. 2013;2013:906813.
  34. Gilman-Sachs A, DuChateau BK, Aslakson CJ, Wohlgemuth GP, Kwak JY, Beer AE, et al. Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions. Am J Reprod Immunol. 1999;41(1):99-105.
  35. Plaisier M, Dennert I, Rost E, Koolwijk P, van Hinsbergh VW, Helmerhorst FM. Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions. Hum Reprod. 2009;24(1):185-97.
  36. Banerjee P, Ghosh S, Dutta M, Subramani E, Khalpada J, Roychoudhury S, et al. Identification of key contributory factors responsible for vascular dysfunction in idiopathic recurrent spontaneous miscarriage. PLoS One. 2013;8(11):e80940.
  37. Col-Madendag I, Madendag Y, Altinkaya SÖ, Bayramoglu H, Danisman N. The role of VEGF and its receptors in the etiology of early pregnancy loss. Gynecol Endocrinol. 2014;30(2):153-6.
  38. Lash GE, Innes BA, Drury JA, Robson SC, Quenby S, Bulmer JN. Localization of angiogenic growth factors and their receptors in the human endometrium throughout the menstrual cycle and in recurrent miscarriage. Hum Reprod. 2012;27(1):183-95.
  39. Almawi WY, Saldanha FL, Mahmood NA, Al-Zaman I, Sater MS, Mustafa FE. Relationship between VEGFA polymorphisms and serum VEGF protein levels and recurrent spontaneous miscarriage. Hum Reprod. 2013;28(10):2628-35.
  40. Amirchaghmaghi E, Rezaei A, Moini A, Roghaei MA, Hafezi M, Aflatoonian R. Gene expression analysis of VEGF and its receptors and assessment of its serum level in unexplained recurrent spontaneous abortion. Cell J. 2015;16(4):538-45.
  41. Muttukrishna S, Swer M, Suri S, Jamil A, Calleja-Agius J, Gangooly S, et al. Soluble Flt-1 and PlGF: new markers of early pregnancy loss? PLoS One. 2011;6(3):e18041.
  42. Fernandes da Silva MO, Elito J Jr, Daher S, Camano L, Fernandes Moron A. Association of serum levels of vascular endothelial growth factor and early ectopic pregnancy. Clin Exp Obstet Gynecol. 2013;40(4):489-91.
  43. Roussev RG, Dons'koi BV, Stamatkin C, Ramu S, Chernyshov VP, Coulam CB, et al. Preimplantation factor inhibits circulating natural killer cell cytotoxicity and reduces CD69 expression: implications for recurrent pregnancy loss therapy. Reprod Biomed Online. 2013;26(1):79-87.
  44. Miko E, Manfai Z, Meggyes M, Barakonyi A, Wilhelm F, Varnagy A, et al. Possible role of natural killer and natural killer T-like cells in implantation failure after IVF. Reprod Biomed Online. 2010;21(6):750-6.
  45. Kalu E, Bhaskaran S, Thum MY, Vishwanatha R, Croucher C, Sherriff E, et al. Serial estimation of Th1:th2 cytokines profile in women undergoing in-vitro fertilization-embryo transfer. Am J Reprod Immunol. 2008;59(3):206-11.
  46. Vodolazkaia A, Yesilyurt BT, Kyama CM, Bokor A, Schols D, Huskens D, et al. Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers. Fertil Steril. 2016;105(4):988-96.
  47. Sergent F, Hoffmann P, Brouillet S, Garnier V, Salomon A, Murthi P, et al. Sustained Endocrine Gland-Derived Vascular Endothelial Growth Factor Levels Beyond the First Trimester of Pregnancy Display Phenotypic and Functional Changes Associated With the Pathogenesis of Pregnancy-Induced Hypertension. Hypertension. 2016;68(1):148-56.