1. Multinucleation per se is not always sufficient as a marker of abnormality to decide against transferring human embryos 
Shu Hashimoto, Ph.D., Tatsuya Nakano, M.S., Kazuo Yamagata, Ph.D., Masayasu Inoue, M.D., Ph.D., Yoshiharu Morimoto, M.D., Ph.D., Yoshiharu Nakaoka, M.D., Ph.D. 
Fertility and Sterility 
Volume 106, Issue 1, Pages e6 (July 2016)
DOI: /j.fertnstert
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Maximum-intensity projections of confocal sections of abnormally cleaved embryos at the first mitosis. Confocal sections were captured every 3.3 μm along the z-axis after injection of RNAs for EGFP-α-tubulin or EB1-EGFP and mRFP1-histone H2B. Numbers on the upper left corner in each panel show elapsed times (in hours:minutes) before or after nuclear envelope breakdown (NEBD). Thirty-five embryos cleaved abnormally at first mitosis (25.2%) (A) with or (B) without showing multipolar spindle formation after chromosome congression and formed MN. (A) This zygote was generated using intracytoplasmic sperm injection. A tripolar spindle was formed by 2.5 hours after NEBD and chromosome congression. Chromosomes were separated into three daughter cells with multiple nuclei. (B) After NEBD, chromosomes were separated without showing chromosome congression in four zygotes (11%). Two spindles formed separately between 0:30 and 2:45. The chromosomes were allocated without chromosome congression at 3:00. One zygote cleaved to four cells, of which three showed multinucleation.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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3. Figure 2
Maximum-intensity projections of confocal sections of embryos with multinucleation: a model of normal cytokinesis of cells with multinucleation. Microtubules orient toward the chromosomes after nuclear envelope breakdown (NEBD) and link with their kinetochores. As a result, all chromosomes involved in multinucleation undergo congression, and a bipolar spindle is formed. The chromosomes are divided equally into two daughter cells. Magnified images of the open squares are shown at upper left corners in each panel. Numbers at the lower left corners show elapsed time (hours:minutes) before or after NEBD. (A) A blastomere with micronuclei underwent normal cleavage and allocated its chromosomes to two daughter cells. Two centrosomes (green spheres) present between times −0:45 and 0:00. Orange arrowheads indicate micronuclei between −0:45 and 0:00, and chromosomes derived from micronuclei at 0:15 and 0:45. The microtubules were oriented toward the chromosomes and were captured by kinetochores. A bipolar spindle was formed at 1:45. The chromosomes were divided equally into the two daughter cells. (B) A blastomere with two nuclei divided normally and allocated its chromosomes to two daughter cells. Two centrosomes (green spheres) were present at time −0:15. Orange arrowheads indicate two nuclei at −0:15 and chromosomes derived from two nuclei at 0:00 and 0:15. Microtubules were oriented toward the chromosomes and linked to kinetochores. A bipolar spindle was formed at 1:15. The chromosomes divided equally into two daughter cells; one formed micronuclei between 2:00 and 3:00. (C) Morphologically “good” blastocyst formation rates from embryos with multinucleation at the two-cell and (D) four-cell stages in the live-cell imaging study. The number in a parenthesis is the number of embryos examined. The chi-square test was used for comparisons between two groups, and the Bonferroni test was applied after analysis of variance for comparisons among more than two groups.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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4. Supplemental Figure 1
Study schema. A live imaging technique was used to determine how the occurrence of multinucleation might be involved in poor developmental competence and whether it would lead to aneuploidy at subsequent developmental stages.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

5. Supplemental Figure 2
(A) Blastulation and (B) morphologically “good” blastocyst formation rates of normally and abnormally cleaved embryos at first mitosis in the live-cell imaging study. The number in parentheses is the number of embryos examined. *P<.05 by chi-square test.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 3
(A) The rates of implantation of embryos that showed normal and abnormal cytokinesis at first mitosis in the clinical study involving embryo transfer. (B) The rates of implantation of embryos with or without multinucleation at the two-cell stage. The number in parentheses is the number of embryos examined. *P<.05 by chi-square test.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions