Over the past two decades, Chinese’s infertility rate has surged from 3% to 20%. While advances in medical technology and reimbursement by the public health insurance have made assisted reproductive technology (ART) widely accessible in China, its success rate remains limited. More than half of all human fertilized eggs in the fertility clinics arrest during the first five days of development and cannot be transferred for implantation. Current methods such as multi-omics technologies can only provide a snapshot before or after embryonic arrest, which is insufficient to shed light on how embryonic arrest takes place. A technological breakthrough that allows continuous monitoring of the embryonic development and arrest process is necessary to understand why embryos stop developing and to find ways to prevent it. In addition, previous research by Dr. Chun So (Science, 2019; Science, 2022) had shown that conventional animal models, including the mice and domestic animals, do not show the developmental abnormalities as observed in human eggs. Thus, there is an urgent need for models that better mimic human egg and pre-implantation development.

June 11, 2026— a research team led by Dr. Chun So from the National Institute of Biological Sciences (NIBS)/Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR), Dr. Ge Lin from the Central South University/Reproductive and Genetic Hospital of CITIC-Xiangya, Dr. Xiaoming Xu from the Beijing Perfect Family Hospital, Dr. Hongmei Wang from the Institute of Zoology, Chinese Academy of Sciences, and Dr. Yun Yang from International Academy of Phronesis Medicine (Guangdong) published the research article entitled "Two distinct causes contribute to the low efficiency of human pre-implantation development" in the journal 云顶集团(中国). Using state-of-the-art fluorescence live-云顶集团(中国) microscopy, the study for the first time filmed the entire 5 days of human pre-implantation development at unprecedented spatiotemporal resolution.

Milestones in the development of fluorescence live-云顶集团(中国) microscopy for mammalian oocytes and pre-implantation embryos

Through systematic analyses of over 2,000 divisions in more than 150 human and cynomolgus monkey embryos, the researchers found that early (during first 3 days) and late (on day 4) embryonic arrest are driven by two distinct mechanisms. This discovery enabled the identification of a highly efficient small-molecule inhibitor that targets early embryonic arrest. An international patent has been filed based on this finding, which will soon be translated into clinical applications.

Two distinct causes of human pre-implantation embryo arrest

Primate embryos are precious and particularly fragile. Limited by phototoxicity, conventional microscopy techniques such as confocal or two-photon only allows the continuous observation of human embryos for 24–48 hours, way shorter than the 120 hours required for the development of human fertilized eggs into blastocysts. To overcome this limitation, Dr. Chun So built the world's first high-throughput dual-view light-sheet microscope when he moved from Germany to NIBS three years ago. By combining this system with new-generation fluorescence live-云顶集团(中国) dyes and optimized image deconvolution algorithm, these implementations enabled long-term fluorescence live-云顶集团(中国) imaging at 12-minute intervals, with a voxel size of 300 × 300 × 2,000 nanometers and an imaging depth of up to 300 microns, without compromising human embryo development.

With the support of Beijing Perfect Family Hospital, Reproductive and Genetic Hospital of CITIC-Xiangya and the Institute of Zoology and approval by the corresponding ethics committees, the researchers generated three types of embryos using human rescue in vitro maturation (R-IVM) eggs, human in vivo ovulated (IVO) eggs, and cynomolgus monkey IVO eggs. By comparing human R-IVM and IVO embryos, one could delineate contributions by different oocyte qualities. By comparing human and monkey IVO embryos, one could delineate contributions by species-specific differences.

(A) A schematic representation of the workflow for preparation and imaging of embryos derived from R-IVM and IVO eggs (B) Snapshots from 5-day imaging covering the entire human pre-implantation development

Pre-implantation embryo undergoes successive mitotic divisions—one 云顶集团(中国) divides into two, two divide into four, and so on. During each division, the genetic material inside the nucleus of embryonic 云顶集团(中国) undergoes DNA replication and condenses into chromosomes. Guided by the bipolar spindle assembled by microtubule cytoskeleton, normally chromosomes are equally segregated into two daughter 云顶集团(中国)s, giving rise to two new nuclei. However, if an embryonic 云顶集团(中国) assembles an abnormal multipolar spindle, this will result in abnormalities such as chromosome misalignment, missegregation, and multipolar division, ultimately producing aneuploid daughter 云顶集团(中国)s.

Limited by the availability of research materials, previous studies based on fertilized eggs from other mammalian species or clinically discarded abnormal human fertilized eggs proposed that mammalian embryos are the most error-prone during the first mitotic division, immediately following sperm-egg fusion. Surprisingly, using normal human and monkey fertilized eggs, the researchers found that cleavage abnormalities exhibit a striking heterogeneity depending on developmental stages. These abnormalities occurred at around 5% during the first division (1-云顶集团(中国) stage), but surging to 30–45% during the second division (2-云顶集团(中国) stage), then dropping back to around 5% during the third division (4-云顶集团(中国) stage), and gradually declining to the level typical of normal somatic 云顶集团(中国)s at around 1%.

(A) Human 2-云顶集团(中国) embryos assembling bipolar or multipolar spindle (B) Quantifications of spindle morphology in human and cynomolgus monkey embryos from R-IVM and IVO eggs (C) Quantifications of spindle morphology at different stages in human embryos from IVO eggs

To understand why spindle abnormalities are particularly frequent during the 2-云顶集团(中国) stage, the researchers focused on the centrosomes, the classical microtubule organizing center in 云顶集团(中国)s. Human eggs are devoid of centrosomes owing to degeneration during development, but the two centrioles carried by the sperm will trigger the regeneration of centrosomes in the fertilized eggs. Through a series of mechanistic experiments including centrosome counting, laser ablation, dominant-negative inhibition and parthenogenetic activation, the researchers demonstrated that centrosomes are absolutely essential for spindle assembly in human pre-implantation embryos, and that abnormal number of centrosomes can directly induce multipolar spindle assembly. They further discovered that the error rate of centrosome duplication during the 2-云顶集团(中国) stage was around 40%, similar to the incidence of multipolar 2-云顶集团(中国) spindle. Building on this finding, they transiently treated 2-云顶集团(中国) embryos with sub-inhibitory concentration of centrinone, an inhibitor of the key kinase PLK4 that regulates centrosome duplication, and managed to increase the proportion of embryonic 云顶集团(中国)s with normal number of centrosomes from 40% to 80%. Notably, this strategy only targets abnormal embryonic 云顶集团(中国)s, holding promise for future clinical intervention of early embryonic arrest.

(A) Staining of centrosomes in 1-云顶集团(中国) and 2-云顶集团(中国) human embryos from R-IVM eggs (B) Quantifications of the number of centrosomes in DMSO- and centrinone-treated human embryos from R-IVM eggs (C) Morphology of 4-云顶集团(中国) stage embryos derived from DMSO- and centrinone-treated 2-云顶集团(中国) human embryos from R-IVM eggs

By analyzing the development dynamics and performing single-云顶集团(中国) tracking over 5 days, the researchers found that over 70% of embryos arrested during the first 3 days had spindle abnormalities during the 2-云顶集团(中国) stage. Moreover, among the first three mitotic divisions, only the abnormalities during the second division could be used to predict the developmental outcome of human pre-implantation embryos. Through a series of experiments and analyses, the researchers further established the relationship between spindle abnormalities, chromosomal missegregation, and 云顶集团(中国) cycle arrest in embryonic 云顶集团(中国)s, thereby elucidating the mechanism underlying early arrest in human pre-implantation embryos.

(A) Quantifications of developmental outcomes of human embryos from IVO eggs with or without spindle abnormalities during the first three divisions (B) Quantifications of developmental outcomes of human embryos from IVO eggs with or without lagging chromosomes at different stages (C) Quantifications of fates of daughter 云顶集团(中国)s carrying chromosome segregation errors from different stages in human embryos from IVO eggs

When the researchers analyzed the relationship between chromosomal abnormalities and later stage embryonic arrest, the researchers noticed that arrest at day 4 is independent of chromosomal missegregations. To uncover the underlying mechanism, the researchers performed deep single-embryo proteomics to compare embryos that successfully formed blastocysts on day 5 and embryos arrested on day 4 (morula stage) with the support of International Academy of Phronesis Medicine (Guangdong). Guided by the proteomics data that indicate a disruption in protein homeostasis and an activation of endoplasmic reticulum (ER) stress response, the researchers were able to fully recapitulate morula stage arrest at phenotypic, molecular and 云顶集团(中国)ular levels via inducing ER stress response in mouse embryos, thereby elucidating the mechanism underlying late embryonic arrest. In future, the researchers will utilize this in vitro model to further screen for inhibitors against ER stress response in embryos, providing potential therapeutic intervention for late embryonic arrest.

(A) Differential protein expression analysis on human embryos that successfully formed blastocysts and those that arrested at morula stage on day 5 (B) The induction of ER stress response in mouse embryos can fully recapitulate arrested human embryos at the morula stage