EXPOSURE OF HUMAN BLASTOCYSTS TO SPECIFIC GROWTH FACTORS BASED ON RECEPTOR PRESENCE IMPROVES EPIBLAST FORMATION IN EXTENDED EMBRYO CULTURE

Abstract

Embryos are exposed to multiple growth factors during pre-implantation development in vivo. However, inclusion of a variety of growth factors in embryo culture medium has resulted in disparate results between studies and across species, making it difficult to ascertain the potential efficacy of growth factors to improve human embryo culture. Our objective was to identify the presence or absence of specific growth factor receptors in human blastocysts using western blot analysis, and then to determine the effect of exposing embryos to the growth factors for which receptors are present on embryo development and viability. Research Study Western blots were conducted using the Protein-Simple Jess system, with detection of each putative growth factor receptor assessed in a pool of two human blastocysts as well as a positive control (human granulosa cells). Due to the paucity of human zygotes that are available for such research, we used vitrified human blastocysts donated for research, exposing blastocysts to growth factors for 48 hrs prior to placement into an extended embryo culture system (WIRB study # 1179872). After warming, day (D) 5 human blastocysts (n=35) were cultured in the presence (GF) or absence (control) of selected growth factors. After 48 hrs (D7), the zona pellucidae were removed prior to placing blastocysts on fibronectin coated 8 well chambered microslides for extended culture. Embryo outgrowth area was measured on D10, D11, and D12, at which time embryos were fixed and stained with DAPI and antibodies against F-actin and the pluripotency marker POU5F1 to determine the number of epiblast cells and total cells using 3D spinning disk confocal microscopy. Data was analyzed using t-test. Of the eleven growth factor receptors analyzed, six were found to be present in human blastocysts. Receptors that were not detected included EGFR, ACVR1, ACVR2, IGF1R, and TGFβR2. There were no differences in embryo outgrowth area on D10, D11, or D12 (control, 0.206 ± 0.053 mm2; GF, 0.234 ± 0.046 mm2). More embryos cultured with chosen growth factors formed an epiblast (control, 35.7%; GF, 84.6%; p=0.03). However, there were no differences in the number of epiblast cells as an average of all embryos (control, 27.8 ± 15.4; GF, 38.8 ± 9.4) or as an average of only those embryos with epiblast cells (control, n=5, 77.8 ± 34.3; GF, n=11, 45.9 ± 9.6). There was also no difference in total cell number (control, 344.1 ± 116.0; GF, 413.4 ± 116.8) in the D12 embryo outgrowths. In summary, inclusion of growth factors in embryo culture medium at the blastocyst stage, chosen based upon receptor presence, increased the ability of the embryos to form an epiblast suggesting that the presence of these growth factors increased embryo viability. Future work will examine the effect of these growth factors during preimplantation embryo culture on blastocyst development and quality prior to embryo transfer.