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Culturing a human embryo for 14 days – what have we learned?

16 May 2016
By Dr Helen O'Neill
Embryology, IVF and Reproductive Genetics Group, Institute for Women's Health,  University College London
Appeared in BioNews 851

Understanding the developmental events that take place following fertilisation has, in recent decades, been driven largely by the desire to improve fertilisation, pregnancy and delivery rates in the clinical IVF setting. Much of this work has relied on the use of mouse embryos, which in the early stages of development appear morphologically similar to human embryos. However, species-specific differences in terms of developmental timing and molecular expression patterns have restricted our true understanding of early human development. 

Following fertilisation, a zygote develops into a blastocyst with two cell lineages: the trophectoderm and the inner cell mass. Within the reproductive tract, the blastocyst is guided down the fallopian tube and starts to implant into the uterus around day seven. The uterus is only receptive for a short time, and the molecular basis for this permissive window is poorly understood. Implantation failure is a major cause of early pregnancy loss, and its understanding is crucial if treatments are to be made available.

Until now, the human pre-implantation period has been solely studied in vitro, but only up until the day when the embryo must be transferred to the uterus in the hopes of a successful pregnancy. In the days following, from day seven onwards, the morphological and cellular events that take place have not been characterised.

Attempts to understand this peri-implantation period in humans have relied on the use of embryos grown in culture conditions that attempt to mimic the uterine environment. However, this work, where human blastocysts were co-cultured with uterine (endometrial) cells, only supported development up to a maximum of nine days (1).

Recent work published in Nature (2) and Nature Cell Biology (3) from Professor Zernicka-Goetz and her team at the University of Cambridge, and Professor Brivanlou at The Rockefeller University, New York, applied previously developed culture techniques using mouse embryos (4, 5) to human embryos to push the record of in vitro culture to 13 days (see BioNews 850).

It has long been assumed that the cellular organisation of a blastocyst, which occurs after implantation, relies on maternal tissues and hormonal signals. This research, however, replaced the use of a maternal layer of uterine cells with a 3D gel matrix to enable outward growth of the embryo. Surprisingly, the transition of cells from pre- to post-implantation occurred in the absence of the maternal uterine environment. This self-organising capacity of the human embryo has never been witnessed before and gives insight into the autonomous ability of the embryo to grow outside of the mother, when cultured in appropriate nutrients.

Currently the debate still stands on whether it is best to transfer embryos cultured for three or five days in an IVF cycle. The information in these latest studies could shed light on better practice for IVF culture conditions by elucidating the unknown genetic and chemical signals needed by the embryo at different times.

The teams also witnessed the formation of a previously uncharacterised cluster of cells as differentiation continued. This new cell population comprised upwards of 5–10 percent of the whole embryo at one point. These cells did not share a genetic expression profile with any other previously studied organism, and they display features unique to human development. They observed that cell sorting of the epiblast and primitive endoderm (cell lineages derived from the inner cell mass) also occurs much later in humans than it does in mice, closer to the time of implantation. This further highlights the disparity between what we can hope to learn from mouse embryonic studies compared with human embryonic studies.

What are the limitations? Is there a rush to change the law?

While this research brings us closer than ever before to understanding this period of embryonic development, culturing in vitro does not fully represent what happens in vivo. We have seen that making assumptions about developmental time points and pathways based on other model organisms is not sufficient, and we must also be careful not to make assumptions about refined experimental culture conditions.

Assisted reproductive technologies have long pushed the parameters of procreation; indeed, the embryos used in this research had already been subject to vitrification (a method of fast freezing), a thawing protocol that attempts to allow re-expansion of an embryo that had been deliberately collapsed (to ensure better freezing), followed by multiple culture conditions and media changes. This may cause a developmental delay and may induce irregular expression profiles in these embryos.

Furthermore, successful hatching of the embryo from the zona pellucida is a prerequisite for implantation in the uterus. Assisted zona hatching was introduced in IVF programmes to encourage the natural process of hatching (6). These embryos were subject to a chemical which removes the zona pelllucida.

Not only has the scientific record been pushed, but now the legal boundary for keeping a human embryo alive in culture is also being tested. The 14-day rule was introduced to the UK in 1984 and became law as part of the HFE, Human Fertilisation and Embryology, Act in 1990. It was designed to act as an ethical boundary, rather than a practical one, as human embryos had previously never survived in culture for longer than seven days. Fourteen days marks the time when gastrulation begins and represents the last developmental time point that the embryo can split to form twins. This time point was therefore assigned relevance not in terms of the neural connectivity of the embryo, but in terms of its formal assignment of being an 'individual'. Many scientists and ethicists still debate the time point at which an embryo should become protected in the eyes of the law.

The constraints of human embryonic research have never before been influenced by the 14-day rule. In fact, the authors of this study themselves suggest a limitation to their two-dimensional culture environment, as outgrowth of the embryo was no longer supported prior to termination of the experiments at 13 days. Research in this field has been restricted in the past because of technical obstacles in analysing individual cells in a given embryo. More problematic still is the inconsistent quality of human embryos that have been donated by IVF patients.

Many would like to see a review of the rules in this area to enable a better understanding of human embryo development. However, others follow the mantra of Danish philosopher Søren Kierkegaard, who said: 'The more a person limits himself, the more resourceful he becomes.' Professor Zernicka-Goetz herself has said that she is not calling for a change in the law, although she added that she would contribute to the debate if there were one. Undoubtedly, that discussion will require input from ethicists and scientists alike.

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