Mitotic spindle disruption in human preimplantation embryos activates the spindle assembly checkpoint but not apoptosis until Day 5 of development.
Is the spindle assembly checkpoint (SAC) active during human preimplantation development?



Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5.



Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking.



In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation.



We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH.



All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells.



Not applicable.



This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care.



Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control.



This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. The authors declare no competing financial interests.
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium.|Institute of Molecular Cancer Research, University of Zurich (UZH), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
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Jacobs
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium.|Centre for Reproductive Medicine, Universitair Ziekenhuis UZ Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
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Van de Velde
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090Brussels, Belgium.
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De Paepe
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090Brussels, Belgium.
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Sermon
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090Brussels, Belgium.
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Spits
D017209:Apoptosis / Q000187:drug effects; D001755:Blastocyst; D002869:Chromosome Aberrations / Q000187:drug effects*; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000187:drug effects* / Q000235:genetics; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects*; D015739:Nocodazole / Q000494:pharmacology*; D011247:Pregnancy; D008941:Spindle Apparatus / Q000187:drug effects* / Q000235:genetics / Q000473:pathology; D059008:Time-Lapse Imaging; D050257:Tubulin Modulators / Q000494:pharmacology*
D050257:Tubulin Modulators; D015739:Nocodazole
aneuploidy; apoptosis; checkpoint uncoupling; human preimplantation embryo; metaphase arrest; spindle assembly checkpoint