Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
1115–1121
Year published:
DOI:
doi:10.1038/nbt.1686
Received
Accepted
Published online

Abstract

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.

At a glance

Figures

  1. Experimental plan.
    Figure 1: Experimental plan.

    We tracked the development of 242 two-pronucleate stage embryos in four experimental sets (containing 61, 80, 64 and 37 embryos, respectively). In each set of experiments, human zygotes were thawed on day 1 and cultured in small groups on multiple plates. Each plate was observed independently with time-lapse microscopy under dark-field illumination on separate imaging stations. At ~24 h intervals, one plate of embryos was removed from the imaging system and collected as either single embryos or single cells (blastomeres) for high-throughput qRT-PCR gene expression analysis. Each plate typically contained a mixture of embryos that reached the expected developmental stage at the time of harvest (termed 'normal') and those that were arrested or delayed at earlier development stages, or fragmented extensively (termed 'abnormal'). Gene expression analysis was carried out on single intact embryos or on single blastomeres of dissociated embryos. One hundred of the 242 embryos were imaged until day 5 or 6 to monitor blastocyst formation.

  2. Abnormal embryos exhibit abnormal cytokinesis and mitosis timing during the first divisions.
    Figure 2: Abnormal embryos exhibit abnormal cytokinesis and mitosis timing during the first divisions.

    (a) The developmental time line of a healthy human preimplantation embryo. Scale bar, 50 μm. (b) The distribution of normal and arrested embryos among samples that were cultured to day 5 or 6. (c) Cytokinesis duration was measured from the appearance of a cleavage furrow to complete daughter-cell separation during the first division. Time between the first and second mitoses was measured from the completion of the first mitosis to the appearance of cleavage furrow of the second mitosis. Synchronicity of the second and third mitoses was defined as the time between the appearance of the cleavage furrows of the second and third mitoses. (d) Normal embryos followed strict timing in cytokinesis and mitosis during early divisions, before EGA begins. Out of the 100 embryos imaged to day 5 or 6, six were excluded from subsequent image analysis due to technical issues (e.g., inability to track identity after media change, or loss of image focus). Raw data for this plot are included as Supplementary Data Set 1, and additional views can be seen in Supplementary Figure 2. (e) Normal cytokinesis (first row) was typically completed in 14.3 ± 6.0 min in a smooth, controlled manner. In the mild phenotype (second row), the cytokinesis mechanism appears normal although it is slightly prolonged. In the severe phenotype (third row), a one-sided cytokinesis furrow is formed, accompanied by unusual ruffling of cell membranes for a prolonged period of time. Cytokinesis was defined by the first appearance of the cytokinesis furrow (arrows) to the complete separation of daughter cells. Imaging was also performed on a subset of triploid embryos (fourth row), which exhibited a distinct phenotype of dividing into three cells in a single event. Scale bar, 50 μm. (f) Embryos that underwent abnormal development and behavior (right) would occasionally appear morphologically similar to normal embryos (left) at the time of sample collection. In this particular case, time-lapse video data showed that what appeared to be a six to eight-cell embryo (right) was in fact the product of a highly aberrant cell division (Supplementary Video 10). Thus, the correlated imaging data served to ensure the accuracy of sample selection and identification for the gene expression analysis.

  3. Automated image analysis confirms the utility of the imaging parameters to predict blastocyst formation.
    Figure 3: Automated image analysis confirms the utility of the imaging parameters to predict blastocyst formation.

    (a) Results of tracking algorithm for a single embryo. Images were captured every 5 min, and only a select group is displayed. The top row shows frames from the original time-lapse image sequence, and the bottom row shows the overlaid tracking results. (b) Set of 14 embryos that were analyzed (Supplementary Video 6). One embryo was excluded as it was floating and out of focus. (c) Comparison of image analysis by a human observer and automated analysis of the duration of cytokinesis (top) and of the time between first and second mitoses (bottom). There is excellent agreement between the two methods for embryos that reached the blastocyst stage with good morphology. The few cases of disagreement occurred mostly for abnormal embryos and were caused by unusual behavior that is difficult to characterize by both methods. The gray shade region shows the window for blastocyst prediction. The two methods agreed on blastocyst prediction except in the case of embryo 10, which was predicted as abnormal by the automated method and normal by the manual method. (d) Comparison of blastocysts with good (top) and bad (bottom) morphology.

  4. Distinct gene expression profiles of developmentally delayed or arrested embryos.
    Figure 4: Distinct gene expression profiles of developmentally delayed or arrested embryos.

    (a) An arrested 2-cell embryo that showed abnormal membrane ruffling during the first cytokinesis had significantly (P < 0.05) reduced expression level of all cytokinesis genes tested. Scale bar, 50 μm. (b) An arrested 4-cell embryo that underwent aberrant cytokinesis with a one-sided cytokinesis furrow and extremely prolonged cytokinesis during the first division showed lower expression of ANLN and ECT2. Scale bar, 50 μm. (c) The average expression level of 52 genes from six abnormal 1- to 2-cell embryos and five normal 1- to 2-cell embryos were plotted in a radar graph on a logarithmic scale. Arrested embryos in general expressed less mRNA than normal embryos, with genes related to cytokinesis, RNA processing and miRNA biogenesis most severely affected. Genes highlighted in orange with an asterisk indicate a statistically significant difference (P < 0.05) between normal and abnormal embryos as determined by the Mann-Whitney test.

  5. Gene expression analysis of single human embryos and blastomeres.
    Figure 5: Gene expression analysis of single human embryos and blastomeres.

    (a) Genes analyzed in human embryos are defined by four distinct ESSPs. Relative expression level of an ESSP was calculated by averaging the expression levels of genes with similar expression patterns. (b) The ratio of maternal to embryonic genes in embryos changes during preimplantation development (left). Some embryos contained blastomeres of different developmental ages (right). The expression levels of embryonic and maternal programs were calculated by averaging the relative expression of ten ESSP1 and ten ESSP2 markers, respectively.

  6. Proposed model for human embryo development.
    Figure 6: Proposed model for human embryo development.

    Human embryos begin life with a set of oocyte RNAs inherited from the mother. After fertilization, a subset of maternal RNAs specific to the egg (ESSP1) must be degraded as the transition from oocyte to embryo begins. As development continues, other RNAs are partitioned equally to each blastomere (ESSP4). At EGA, ESSP2 genes are transcribed in a cell-autonomous manner. During the cleavage divisions, embryonic blastomeres may arrest or progress independently. 'Feature extraction' indicates the three imaging parameters for predicting successful development to the blastocyst stage: cytokinesis, the time between 1st and 2nd mitoses, and the time between 2nd and 3rd mitoses.

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Author information

  1. These authors contributed equally to this work.

    • Connie C Wong &
    • Kevin E Loewke

Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University, Stanford, California, USA.

    • Connie C Wong,
    • Kevin E Loewke &
    • Renee A Reijo Pera
  2. Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, California, USA.

    • Connie C Wong,
    • Kevin E Loewke,
    • Barry Behr &
    • Renee A Reijo Pera
  3. Department of Mechanical Engineering, Stanford University, Stanford, California, USA.

    • Kevin E Loewke
  4. Reproductive Medicine Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • Nancy L Bossert &
    • Christopher J De Jonge
  5. Stanford Photonics Research Center, Department of Applied Physics, Stanford University, Stanford, California, USA.

    • Thomas M Baer
  6. Present address: Auxogyn, Inc., Menlo Park, California, USA.

    • Kevin E Loewke

Contributions

C.C.W. and K.E.L. performed and designed experiments, analyzed data and assisted in writing and editing of the manuscript. K.E.L. designed cell tracking algorithms. N.L.B. assisted in performing the experiments. B.B., N.L.B. and C.J.D.J. assisted in analyzing data and editing the manuscript. T.M.B. and K.E.L. designed and built the imaging instrumentation. T.M.B. and R.A.R.P. designed experiments, interpreted results and assisted in writing and editing the manuscript.

Competing financial interests

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.

Corresponding author

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Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (1 MB)

    Supplementary Tables 1,2 and Supplementary Figs. 1–10

Movies

  1. Supplementary Video 1 (8 MB)

    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.

  2. Supplementary Video 2 (24 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.

  3. Supplementary Video 3 (28 KB)

    Video accompaniment to Figure 1e (second panel). Some embryos underwent a slightly delayed but otherwise morphologically normal cytokinesis.

  4. Supplementary Video 4 (416 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.

  5. Supplementary Video 5 (296 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.

  6. Supplementary Video 6 (6 MB)

    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.

  7. Supplementary Video 7 (11 MB)

    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.

  8. Supplementary Video 8 (64 KB)

    Video accompaniment to Figure 3a. Abnormal membrane ruffling was observed during the first cytokinesis of this arrested 2-cell embryo.

  9. Supplementary Video 9 (600 KB)

    Video accompaniment to Figure 3b. This arrested 4-cell embryo underwent a severely abnormal cytokinesis during its first division.

  10. Supplementary Video 10 (7 MB)

    Video accompaniment to Supplementary Figure 1f. Video microscopy data aided in the identification of abnormal embryos (bottom) from normal embryos (top).

Excel files

  1. Supplementary Data Set 1 (52 KB)

    Raw data used to generate Figure 1d.

  2. Supplementary Data Set 2 (60 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.

  3. Supplementary Data Set 3 (52 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.

  4. Supplementary Data Set 4 (44 KB)

    Taqman probes used for qRT-PCR analysis.

  5. Supplementary Data Set 5 (156 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.

  6. Supplementary Data Set 6 (300 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.

  7. Supplementary Data Set 7 (160 KB)

    High throughput qRT-PCR data set 3. 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.

Additional data