Complete zona pellucida removal from vitrified-warmed human blastocysts facilitates earlier in-vitro attachment and outgrowth Satoshi Ueno, Kenji Ezoe,

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Complete zona pellucida removal from vitrified-warmed human blastocysts facilitates earlier in-vitro attachment and outgrowth Satoshi Ueno, Kenji Ezoe, Akiko Yabuuchi, Kazuo Uchiyama, Tadashi Okimura, Takashi Okuno, Tamotsu Kobayashi, Keiichi Kato Reproductive BioMedicine Online Volume 33, Issue 2, Pages 140-148 (August 2016) DOI: 10.1016/j.rbmo.2016.05.007 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Total zona pellucida removal of vitrified–warmed blastocysts. (A) Expanded blastocyst after warming procedure. (B) Blastocyst shrinkage by high osmolality solution and pipetting. (C) Partial removal of zona pellucida from blastocyst by laser. (D and E) Complete zona pellucida removal by gentle pipetting. (F) Blastocyst with zona pellucida completely removed. Arrowhead indicates the blastocyst. Scale bars represent 100 µm. Reproductive BioMedicine Online 2016 33, 140-148DOI: (10.1016/j.rbmo.2016.05.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Blastocyst adhesion and outgrowth after assisted hatching. (A) Cumulative blastocyst adhesion rate at 0, 12, 18, 24, 36, 48, 72, 96 h after assisted hatching treatment. (B) Time required for adhesion. (C) Outgrowth speed of blastocysts. Error bars represent the standard error of the mean. Bars labelled with asterisk are statistically significantly different from each other (P < 0.05). Due to a low number of hatched and adhered blastocysts (n = 3) in the intact group, adhesion time and blastocyst outgrowth speed in the Intact group were not statistically compared with those of the P- or C-removal groups. Reproductive BioMedicine Online 2016 33, 140-148DOI: (10.1016/j.rbmo.2016.05.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Integrin mRNA expression in blastocysts after assisted hatching. (A) ITGA5 mRNA expression in blastocyst after assisted hatching treatment. (B) ITGB1 mRNA expression in blastocyst after assisted hatching treatment. Error bars represent the standard error of the mean. Bars labelled with distinct letters are statistically significantly different from each other (P < 0.05). Reproductive BioMedicine Online 2016 33, 140-148DOI: (10.1016/j.rbmo.2016.05.007) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions