Culture and vitrification of human preembryos

Despite improvements in stimulation protocols, culture media formulations and laboratory protocols, the success rates in human IVF remain disappointingly low. The ability to successfully cryopreserve supernumerary embryos in a given IVF cycle without losing significant embryo viability is essential to maximize the cumulative benefit of a given treatment cycle. Therefore, studies on culture, cryopreservation and gene expression of human embryos fertilized in vitro were performed. In these studies the impact of culture media on fertilization of human oocytes in vitro was investigated. Furthermore, the impact of growth factor supplementation to in vitro culture media and embryo survival and cryodamage after vitrification were studied. Using in situ hybridization and immunohistochemistry methods, the expression of genes in the human Fallopian tube, endometrium, and pre-implantation embryos and in human embryonic stem cells (hES) cells was studied. The findings can be summarized as follows: in vitro culture media has impact on normal fertilization. Supplementation of growth factors to in vitro culture media implicates a physiological role in regulating pre-implantation development. Vitrification of embryos is an effective way of cryopreservation. In situ hybridization, immunohistochemical and matrix assisted laser desorption/ionization time of flight mass spectrometry methods are versatile tools in reproductive medicine research. These findings will help to identify markers for embryo development and characterisation of hESC. Furthermore, knowledge obtained will give us tools to improve formulations of culture and cryopreservation media, which in turn might increase the overall results in IVF treatment and maximise the usage of hESC. ISBN 978-91-7549-577-4 LIST OF PUBLICATIONS This thesis is based on the following original publications referred to in the text by their Roman numerals. I. Hambiliki F, Sandell P, Yaldir F, Stavreus-Evers A. A prospective randomized sibling-oocyte study of two media systems for culturing cleavage-stage embryos-impact on fertilization rate. J Assist Reprod Genet, 2011, 28 (4), 335-41. II. Hambiliki F, Hanrieder J, Bergquist J, Hreinsson J, Stavreus-Evers , Wånggren K. Glycoprotein 130 promotes human blastocyst development in vitro. Fertil Steril. 2013 May; 99(6):1592-9. III. Kartberg AJ, Hambiliki F, Arvidsson T, Stavreus-Evers A, Svalander P. Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO. Reprod Biomed Online, 2008, 17 (3), 378-84. IV. Hambiliki F, Ström S, Zhang P, Stavreus-Evers A. Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells. J Assist Reprod Genet. 2012 Jun 29. V. Wånggren K, Lalitkumar PG, Hambiliki F, Ståbi B, GemzellDanielsson K, Stavreus-Evers A. Leukaemia inhibitory factor receptor and gp130 in the human Fallopian tube and endometrium before and after mifepristone treatment and in the human preimplantation embryo. Mol Hum Reprod, 2007, 13 (6) 391-7. PUBLICATIONS NOT INCLUDED IN THIS THESIS VI. Hambiliki F, Ljunger E, Karlström PO, Stavreus-Evers A. Hyaluronan-enriched transfer medium in cleavage-stage frozenthawed embryo transfers increases implantation rate without improvement of delivery rate. Fertil Steril. 2010 Oct; 94 (5):1669-73. VII. Zhang P, Zucchelli M, Bruce S, Hambiliki F, Stavreus-Evers A, Levkov L, Skottman H, Kerkelä E, Kere J, Hovatta O. Transcriptome profiling of human pre-implantation development. PLoS One. 2009 Nov 16;4(11):e7844. VIII. Lalitkumar PG, Lalitkumar S, Meng CX, Stavreus-Evers A, Hambiliki F,Bentin-Ley U, Gemzell-Danielsson K. Mifepristone, but not levonorgestrel, inhibits human blastocyst attachment to an in vitro endometrial three-dimensional cell culture model. Hum Reprod. 2007 Nov; 22 (11):3031-7. IX. Zhang P, Dixon M, Zucchelli M, Hambiliki F, Levkov L, Hovatta O, Kere J. Expression analysis of the NLRP gene family suggests a role in human preimplantation development. PLoS One. 2008 Jul 23; 3 (7). X. Nordqvist S, Kårehed K, Hambiliki F, Wånggren K, Stavreus-Evers A, Akerud H. The presence of histidine-rich glycoprotein in the female reproductive tract and in embryos. Reprod Sci. 2010 Oct; 17(10):941-7.