Polyethylene glycated leukemia inhibitory factor antagonist inhibits human blastocyst implantation and triggers apoptosis by down-regulating embryonic AKT  Sujata Lalitkumar, M.D., Nageswara R. Boggavarapu, M.Sc., Judith Menezes, Ph.D., Evdokia Dimitriadis, Ph.D., Jian-Guo Zhang, Ph.D., Nicos A. Nicola, Ph.D., Kristina Gemzell-Danielsson, M.D., Ph.D., Luther P.G. Lalitkumar, Ph.D.  Fertility and Sterility  Volume 100, Issue 4, Pages 1160-1169.e2 (October 2013) DOI: 10.1016/j.fertnstert.2013.06.023 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Flow chart showing the details of the human blastocyst allocation to various treatments in the study. Fertility and Sterility 2013 100, 1160-1169.e2DOI: (10.1016/j.fertnstert.2013.06.023) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Human embryos that were hatched and attached in the control group were exposed to PEG in vehicle in the in vitro three-dimensional endometrial construct. Here they are seen after termination and washing of the construct. (B) Inhibition of LIF by PEGLA led to degeneration of blastocysts in vitro as seen after 5 days of placing the blastocyst onto the endometrial construct. The cell mass is shrunk, leaving a large space between the degenerated cells and the zona of the human blastocyst. (This picture was obtained before washing the construct because washing the construct would remove the unattached blastocysts from the insert.) A healthy blastocyst cannot remain in the stage seen in B after 5 days of it forming. The degenerated blastocyst in B was washed away on termination of the culture. The arrow shows a blastocyst on the endometrial construct. Bar = 50 μm. Fertility and Sterility 2013 100, 1160-1169.e2DOI: (10.1016/j.fertnstert.2013.06.023) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Treatment of endometrial constructs with PEGLA (800 pg/mL) decreased the expression of both LIF (A) and gp130 (B) in epithelial cells compared with the control groups. Fertility and Sterility 2013 100, 1160-1169.e2DOI: (10.1016/j.fertnstert.2013.06.023) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) The control group of human blastocysts expressed the cell survival factor phospho-AKT-1, whereas (B) inhibition of LIF with PEGLA (800 pg/mL) led to the down-regulation of phospho-AKT-1 in the inner cell mass. (C) The blastocysts in the control group showed no statistically significant activation of caspase-3. (D) Inhibition of LIF by PEGLA (800 pg/mL) led to an increase of activated caspase 3 or apoptosis in human blastocysts. (E) Negative control using rabbit IgG. Red = positive phospho-AKT-1/activated caspase 3. Blue = nuclear stain for DAPI. Bar = 20 μm. Fertility and Sterility 2013 100, 1160-1169.e2DOI: (10.1016/j.fertnstert.2013.06.023) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions