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. 2017 Aug 15;7:8299. doi: 10.1038/s41598-017-08266-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Micromanipulation and culture of two-cell mouse embryos to generate monozygotic (MZ) twins. Embryos were held in a micromanipulation medium lacking calcium and phosphate. A holding pipette (right side in the picture of series A) applied negative pressure to hold a two-cell stage embryo at the equatorial region, aligning its cleavage axis with the pipette’s long axis. Using a spiked beveled pipette as a bisection tool (left side in the picture), a slit was made in the zona pellucida (ZP) at the upper pole of the embryo (A1). The upper blastomere was pushed through the slit by gently pressing the bisection pipette against the ZP at the equator, while the pressure in the holding pipette was slightly reduced (A2–A3). The second blastomere was released by pressing the ZP below the equator (A4–A5). Separated blastomeres recovered their round shape within a few minutes after bisection (A6). Upon culture in KSOM(aa) medium, the sister blastomeres cleaved and developed to the four-cell stage (planar conformation, (B1); tetrahedral conformation, (B2); open chain conformation, B3) and further to the blastocyst stage (B4). When MZ blastocysts were transferred as a pair to the same uterine horn, cases were recorded in which two fetuses were found in that horn (B5), suggesting that at least some two-cell embryos were totipotent in both blastomeres. Monozygotic twins formed blastocysts at the same rate (C1), comprising half the number of cells as unmanipulated blastocysts (C3). After transfer to the uterus, blastocysts obtained from bisected and then immediately reunited blastomeres yielded as many fetuses as unmanipulated controls (C2). The error bars associated with the diagram bars are standard deviations. Twin pairs that formed two blastocysts were equally frequent among the sister blastomeres, as were twin pairs that arrested at pre-blastocyst stages (C4).