Environment is vital: development of in vitro mouse embryos lag behind their in vivo counterparts

Figure 1. Graphical abstract: developmental progression of in vitro mouse embryos is consistently delayed in comparison to in vivo embryos. [Illustration by Heather Pollington/Adobe Illustrator].

Significance of study

For placental animals, the female reproductive system provides an optimal environment for a growing embryo. But, how essential is this environment? Dr. Huanhuan Yang from the University of Manchester asked this very question, comparing the early developmental trajectory of in vivo and in vitro mouse embryos. His findings highlight the importance of the uterine environment for proper embryonic development and show that in vitro development is severely delayed in multiple developmental processes. Previous work has demonstrated that early 2-cell embryos may possess a great amount of resilience to environmental variations^1,2. Consistent with this hypothesis, Dr. Yang observed similar embryonic volume in both in vivo and in vitro embryos at the 2-cell. However, developmental delays observed in in vitro embryos at later stages suggest adjustments in environmental conditions may be required to further assist developmental progression. In addition, this study reveals that continual live imaging may be significantly harmful to the developing embryo.

Introduction

Life. For every being on earth, it begins with the journey of a single cell. For mammals, this journey begins deep in the female reproductive system where a viable egg-cell travels through the fallopian tube and implants into the uterus, making a home to grow and mature until birth. However, successful development hinges on the proper coordination of critical, initial processes in early embryo development.

While navigating down the fallopian tube, a single egg-cell divides to give rise to a 2-celled organism enveloped by a protective shell. Each of these cells continue to divide, exponentially increasing cell numbers in the growing embryo to 4-cells, 8-cells, and beyond, forming a highly complex multicellular organism³. At the 4-cell stage, the embryo undergoes a process called compaction, in which the 4 cells, once loosely floating, now tightly connect to one another^3,4. Upon increasing the number of tightly compacted cells, the embryo then undergoes cavitation, forming a distinct cavity that arranges two separate cell types: one that will form the embryo and the other that will facilitate implantation into the uterus. Like a newborn chick, the embryo then hatches out of the protective shell to implant into the uterine wall⁴.

Failure or delay of these processes have been associated with human infertility and miscarriages^5,6. In vitro fertilization (IVF) has significantly assisted in identifying embryos that may be faltering at a particular stage, allowing for desiring families to successfully achieve a long-awaited conception. In vitro studies, observing embryo development outside the native uterine environment, and continuous live recordings, have also significantly increased our understanding of these crucial, defining steps and the developmental time at which they occur. However, a recently published preprint by Huanhuan Yang at the University of Manchester suggests that in vitro embryo development may be significantly impacted when compared to in vivo embryos of the same stage⁷.

Key findings

Compaction, cavitation, and hatching are delayed in in vitro embryos

Dr. Yang compared the organization and timing of initial developmental processes in in vivo and in vitro mouse embryos. In vivo embryos were collected at various developmental time points providing snapshots of embryo progression while in vitro embryos were continuously recorded for 5 days. In vivo embryos displayed tightly compacted cells before the 8-cell stage, as expected, while in vitro embryos only began to initiate compaction at the 8-cell stage, many taking longer to fully complete the processes. In vitro cavity formation was also delayed, beginning between the 31-35 cell stage, while in vivo cavity initiation began at the 30-32 cell stage. Consistent with previous observations, in vivo embryos began hatching before the 96-cell stage⁴. In contrast, in vitro embryo hatching was substantially delayed, only just beginning around the 98-cell stage, with many embryos becoming trapped within the protective shell and delaying or failing to completely hatch.

In vitro embryos show a slower growth rate and retain a rounded shape

Dr. Yang further examined the overall size of embryos in each group and observed that initially, in vivo and in vitro 2-cell embryos possess a similar volume. However, in vivo embryos grew significantly faster, larger, and displayed a change in shape from a spherical to an oblong appearance as development progressed. In contrast, in vitro embryos retained a rounded form. Consistent with these findings, embryo cell number increased exponentially overtime in in vivo embryos, while in vitro embryos showed progressively less exponential growth, resulting in significantly fewer cells by the time embryo hatching was initiated.

Future directions

In future work, the comparative addition of an in vitro group collected using a snapshot approach, as opposed to live imaging, would provide a valuable comparison to isolate and identify the effects that continual recordings may have on these processes. Thus, it is unknown to what extent embryos created using IVF are affected, as many of these embryos do not experience the constant imaging exposure that the in vitro embryos underwent in this study. However, this comparative study illuminates a great need for a better understanding of the long-term consequences that may arise during interventions such as IVF, and how we might improve upon fertility treatment methods.

 

References

1. Spindle, A. I. Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos. J Exp Zool 203, 483–489 (1978).
2. Yao, C., Zhang, W. & Shuai, L. The first cell fate decision in pre-implantation mouse embryos. Cell Regen 8, 51–57 (2019).
3. Zhu, M. & Zernicka-Goetz, M. Principles of Self-Organization of the Mammalian Embryo. Cell 183, 1467–1478 (2020).
4. Yang, H. Spatiotemporal Frameworks of Morphogenesis and Cell Lineage Specification in Pre- and Peri-Implantation Mammalian Embryogenesis: Insights and Knowledge Gaps from Mouse Embryo. Biology (Basel) 14, 1596 (2025).
5. Coticchio, G. et al. Perturbations of morphogenesis at the compaction stage affect blastocyst implantation and live birth rates. Hum Reprod 36, 918–928 (2021).
6. Agenor, A. & Bhattacharya, S. Infertility and Miscarriage: Common Pathways in Manifestation and Management. Womens Health (Lond Engl) 11, 527–541 (2015).
7. Yang, H. Cross Sectional and Longitudinal Imaging Reveals Spatiotemporal Divergence in Morphogenesis and Cell Lineage Specification between in-vivo and in-vitro Mouse Embryo during Pre- and Peri-implantation. 2025.12.15.691388 Preprint at https://doi.org/10.64898/2025.12.15.691388 (2026).

Tags: development, embryo, in vitro, mouse

doi: https://doi.org/10.1242/prelights.42981

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