We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.
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We thank R. Raja for help with the microarray analysis and early imaging experiments, the members of the Reijo Pera laboratory for technical assistance and discussions, S. Walker for advice regarding the cell tracking algorithm and K. Salisbury for providing K.E.L. with hardware and software resources. We acknowledge funding contributions from the Stanford Institute for Stem Cell Biology and Regenerative Medicine, a generous, anonymous donor and the March of Dimes (6-FY06-326).
This research project was conducted at Stanford University, and at the time of original submission there were no competing financial interests. K.L. is now an employee of Auxogyn, Inc., which has licensed intellectual property resulting from this research. C.W., K.L., N.B., B.B., C.J.D., T.B. and R.R.P. own stock in Auxogyn.
Supplementary Tables 1,2 and Supplementary Figs. 1–10 (PDF 1216 kb)
Video accompaniment to Figure 1a. The development of 15 human zygotes was documented with darkfield time-lapse microscopy. Images were taken at 1 second exposure time every 5 minutes for 6 days. Media was changed on Day 3, resulting in the rearrangement of individual embryo's location. The identity of each embryo was tracked by videotaping the process of sample transfer during media change and sample collection. Among the 15 embryos, 10 developed into a blastocyst and 5 became arrested at different stages of development. Embryo H in this video corresponds to the embryo depicted in Figure 2a. (MOV 8241 kb)
Video accompaniment to Figure 1e (first panel). A normal embryo typically completed cytokinesis in 13.0 +/- 4.2 min in a smooth and controlled manner. (MOV 21 kb)
Video accompaniment to Figure 1e (second panel). Some embryos underwent a slightly delayed but otherwise morphologically normal cytokinesis. (MOV 26 kb)
Video accompaniment to Figure 1e (third panel). In the more severe phenotype, the abnormal embryos often formed a one-sided cytokinesis furrow accompanied by extensive membrane ruffling before finally completing the division, possibly resulting in embryo fragmentation. (MOV 415 kb)
Video accompaniment to Figure 1e (fourth panel). Imaging was also performed on a subset of triploid embryos which exhibited a distinct phenotype of dividing into 3-cells in a single event. (MOV 293 kb)
Video accompaniment to Figure 2a. Results of 2D tracking algorithm for a single embryo. Images are acquired every 5 minutes. The movie shows the most probable model, the original image, the Hessian (principle curvature image), the thresholded Hessian, and the simulated image (which corresponds to the most probable model). The plots on the bottom show the particles, with dots placed at the centers of the cells, before and after re-sampling. (MOV 6607 kb)
Video accompaniment to Figure 2b. 2D tracking for a set of 14 embryos. One embryo was excluded from image analysis since it was floating and out of focus. Once the algorithm is capable of making a prediction of blastocyst, the embryo is labeled with 'viable' for blastocyst or 'non-viable' for non-blastocyst. On day 3 there is a media change that allows the embryos to be culturedto the blastocyst stage. This process was videotaped to assist in maintaining embryo identity. (MOV 11447 kb)
Video accompaniment to Figure 3a. Abnormal membrane ruffling was observed during the first cytokinesis of this arrested 2-cell embryo. (MOV 61 kb)
Video accompaniment to Figure 3b. This arrested 4-cell embryo underwent a severely abnormal cytokinesis during its first division. (MOV 599 kb)
Video accompaniment to Supplementary Figure 1f. Video microscopy data aided in the identification of abnormal embryos (bottom) from normal embryos (top). (MOV 7409 kb)
Raw data used to generate Figure 1d. (XLS 49 kb)
Complete probe list used for each experiment, as well as the corresponding Unigene ID and RefSeq Accession ID of each ABI assay-on-demand probe, as provided on Applied Biosystems' website. (XLS 57 kb)
Comparison of our qRT-PCR gene expression data in 1-cell and 2-cell embryos to the microarray data in human oocytes as described in Kocabas et al. We note that due to the differences in experimental design and data handling, we would only expect qualitative agreement between these 2 data sets. Expression of two genes, AURKA and CCNA1, was also analyzed in a separate report by Keissling et al. (J Assist Reprod Genet (2009) 26:187–195)11; expression of these genes was consistent with our data and that of Kocabas et al. These genes are indicated by an asterisk; overlap between gene sets was minimal due to differences in experimental design. (XLS 49 kb)
Taqman probes used for qRT-PCR analysis. (XLS 41 kb)
High throughput qRT-PCR data set 1. This excel file contains the relative expression values of all samples and genes assayed in the first high throughput qRT-PCR experiment. Samples were named using a 3-part nomenclature: part 1 depicted the developmental stage of the embryo, part 2 indicated the order of the sample collected within its category, and part 3 reflected whether the embryo was collected as a whole embryo or single blastomere. For example, the name '2c-7-1' referred to the 1st blastomere of the 7th 2-cell embryo collected, whereas 'B-10-W' was the 10th blastocyst collected as a whole embryo. (XLS 156 kb)
High throughput qRT-PCR data set 2. This excel file contains the relative expression values of all samples and genes assayed in the second high throughput qRT-PCR experiment. The sample nomenclature scheme was the same as Supplementary Dataset 5. (XLS 300 kb)
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Wong, C., Loewke, K., Bossert, N. et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat Biotechnol 28, 1115–1121 (2010). https://doi.org/10.1038/nbt.1686
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