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
- The unique transcriptome through day 3 of human preimplantation development. Hum. Mol. Genet. 13, 1461–1470 (2004). et al.
- Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332, 459–461 (1988). , &
- Zygotic and embryonic expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote 8, 87–96 (2000). &
- Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol. Reprod. 71, 185–193 (2004). et al.
- DNA methylation pattern in human zygotes and developing embryos. Reproduction 128, 703–708 (2004). , , &
- Preimplantation embryo programming: transcription, epigenetics, and culture environment. Reproduction 135, 141–150 (2008). , &
- A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev. Cell 6, 133–144 (2004). et al.
- RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo. Dev. Biol. 283, 40–57 (2005). &
- Chromosome instability is common in human cleavage-stage embryos. Nat. Med. 15, 577–583 (2009). et al.
- Conception to ongoing pregnancy: the “black box” of early pregnancy loss. Hum. Reprod. Update 8, 333–343 (2002). , &
- Female subfertility. Lancet 360, 151–159 (2002).
- Does severe teratozoospermia affect blastocyst formation, live birth rate, and other clinical outcome parameters in ICSI cycles? Fertil. Steril. 93, 1097–1103 (2010). , , &
- Culture and transfer of viable blastocysts: a feasible proposition for human IVF. Hum. Reprod. 15 (Suppl 6), 9–23 (2000). , &
- Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod. 12, 532–541 (1997). , , &
- Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. Gene 237, 373–383 (1999). , &
- The human cumulus—oocyte complex gene-expression profile. Hum. Reprod. 21, 1705–1719 (2006). et al.
- Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors. Reprod. 135, 635–647 (2008). et al.
- Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod. 9, 1743–1748 (1994). , , , &
- Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro . Hum. Reprod. 17, 407–412 (2002). , , &
- Early embryo cleavage is a strong indicator of embryo quality in human IVF. Hum. Reprod. 16, 2652–2657 (2001). , &
- Kinetic markers of human embryo quality using time-lapse recordings of IVF/ICSI-fertilized oocytes. Reprod. Biomed. Online 17, 385–391 (2008). , &
- Expression profiles of individual human oocytes using microarray technology. Reprod. Biomed. Online 8, 325–337 (2004). et al.
- The transcriptome of human oocytes. Proc. Natl. Acad. Sci. USA 103, 14027–14032 (2006). et al.
- Significance of morphological attributes of the early embryo. Reprod. Biomed. Online 10, 669–681 (2005). et al.
- Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development. Front. Biosci. 12, 3713–3726 (2007). &
- Evidence that human blastomere cleavage is under unique cell cycle control. J. Assist. Reprod. Genet. 26, 187–195 (2009). et al.
- The role of centrosomes in fertilization, cell division and establishment of asymmetry during embryo development. Semin. Cell Dev. Biol. 21, 174–184 (2010). &
- Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature 429, 154 (2004). , , , &
- Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473–478 (2009). et al.
- Patterning of the embryo: the first spatial decisions in the life of a mouse Development 129, 815–829 (2002).
- The first cleavage of the mouse zygote predicts the blastocyst axis. Nature 434, 391–395 (2005). et al.
- Embryology: does prepatterning occur in the mouse egg? Nature 442, E3–4 (2006). , , &
- The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice Curr. Opin. Genet. Dev. 16, 406–412 (2006).
- High rates of embryonic loss, yet high incidence of multiple births in human ART: Is this paradoxical? Theriogenology 57, 87–96 (2002).
- Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil. Steril. 73, 126–129 (2000). , , , &
- Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 73, 1155–1158 (2000). , , , &
- Towards a single embryo transfer. Reprod. Biomed. Online 6, 470–481 (2003). &
- Single blastocyst transfer: a prospective randomized trial. Fertil. Steril. 81, 551–555 (2004). et al.
- Imprinting disorders and assisted reproductive technology. Fertil. Steril. 91, 305–315 (2009). , &
- Epigenetics and assisted reproductive technology: a call for investigation. Am. J. Hum. Genet. 74, 599–609 (2004). &
- Assisted reproduction: the epigenetic perspective. Hum. Reprod. Update 11, 473–482 (2005). &
- Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate. Reprod. Biomed. Online 20, 510–515 (2010). et al.
- Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril. 59, 1202–1207 (1993). et al.
- In vitro development and implantation rates of fresh and cryopreserved sibling zygotes. Obstet. Gynecol. 85, 999–1002 (1995). &
- Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil. Steril. 72, 1049–1054 (1999). , , , &
- Vitrification in assisted reproduction: myths, mistakes, disbeliefs and confusion. Reprod. Biomed. Online 19, 1–7 (2009). , , , &
- Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod. Biomed. Online 4, 146–150 (2002). et al.
- Origin and ploidy of multipronuclear zygotes. Reprod. Fertil. Dev. 11, 273–279 (1999). , &
- Development of the human dispermic embryo (CD-ROM). Hum. Reprod. Update 5, 553–560 (1999). et al.
- Relationship between embryo quality and aneuploidies. Reprod. Biomed. Online 12, 77–82 (2006). et al.
- How good is embryo morphology at predicting chromosomal integrity? When is aneuploidy PGD useful? Fertil. Steril. 84, S98–S99 (2005). et al.
- Aneuploidy rates of human preimplantation embryos in relation to morphology and development. Fertil. Steril. 86, S474 (2006). et al.
- A novel and critical role for Oct4 as a regulator of the maternal-embryonic transition. PLoS ONE 3, e4109 (2008). et al.
- Analysis of relative gene expression data using real time quantitative PCR and the 2-deltaCT Method. Methods 25, 402–408 (2001). &
- Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 0034.1–0034.12 (2002). et al.
- qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19.1–R19.14 (2007). , , , &
- Computational prediction of neural progenitor cell fates. Nat. Methods 7, 213–218 (2010). , , &
- Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008). et al.
- Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods 5, 687–694 (2008). , , &
- Automated cell lineage tracing in Caenorhabditis elegans . Proc. Natl. Acad. Sci. USA 103, 2707–2712 (2006). et al.
- Supplementary Text and Figures (1 MB)
Supplementary Tables 1,2 and Supplementary Figs. 1–10
- 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.
- 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.
- Supplementary Video 3 (28 KB)
Video accompaniment to Figure 1e (second panel). Some embryos underwent a slightly delayed but otherwise morphologically normal cytokinesis.
- 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.
- 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.
- 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.
- 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.
- 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.
- Supplementary Video 9 (600 KB)
Video accompaniment to Figure 3b. This arrested 4-cell embryo underwent a severely abnormal cytokinesis during its first division.
- 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).
- Supplementary Data Set 1 (52 KB)
Raw data used to generate Figure 1d.
- 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.
- 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.
- Supplementary Data Set 4 (44 KB)
Taqman probes used for qRT-PCR analysis.
- 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.