Dutch researchers have found the first evidence that a process of
inactivating the X chromosome during embryo development and
implantation, which was known to occur in mice but unknown in humans,
does, in fact, take place in human female embryos prior to implantation
in the womb.
Ms Ilse van den Berg told the 25th annual meeting of the
European Society of Human Reproduction and Embryology in Amsterdam June
29 that her findings may have implications for the laboratory cultures
that embryos are grown in before transfer to a woman’s womb during
fertility treatment, as well as for embryo stem cell research.
Males and females have two sex chromosomes: X and Y. While females
have two X chromosomes and no Y chromosome, males have one of each. As
the X chromosome is much larger then the Y chromosome, males and females
also differ in their numbers of genes and gene expression. To equalise
this difference in gene expression, females need to silence one X
chromosome in every cell – a process known as X chromosome inactivation
In mice, XCI occurs before embryo implantation when the X chromosome
inherited from the father is turned off, while the maternal X chromosome
remains turned on. As the cells carry on dividing and reach the
blastocyst stage, the cells that will go on to form the placenta
continue to have the paternal X chromosome switched off, but it is
switched back on in the cells that are going to form the inner cell mass
that develops into the foetus.
“It is from these cells that mice embryonic stem cell lines are made;
these ES cells have two active X chromosomes and are capable of
becoming any kind of cell in the body. As soon as the cells are going to
differentiate into any kind of specialised cell, one X chromosome is
turned off again, but this time it is a random process and it can be
either the maternal or paternal X chromosome that is switched off,”
explained Ms van den Berg, who is a PhD student at Erasmus Medical
Centre (Rotterdam, The Netherlands).
If preimplantation XCI in mice fails for any reason, it results in
cell and embryo death.
Due to the difficulties of investigating XCI in human embryos
(because of the shortage of embryos available for research), no one knew
how XCI worked in humans and, in fact, it was thought that this initial
inactivation of the X chromosome before implantation did not happen at
all, and that only the random XCI after implantation occurred.
However, Ms van den Berg and her colleagues have now found the first
evidence that XCI does occur in pre-implantation embryos, indicating
that this mechanism of compensating for gene dosages has remained
basically unchanged throughout evolution and is probably the same in all
mammals that have their young attached to a placenta in the womb – from
mice to humans.
The researchers looked at human embryos that had been donated for
scientific research by couples undergoing fertility treatment. They
studied them at three stages of their development: after the embryo had
divided into eight cells, the morula stage (a solid cluster of
approximately 16 cells) and the blastocyst stage (about five days after
fertilisation when the embryo’s cells have started to differentiate into
different cell layers).
They used probes designed for detecting a gene called XIST (X
chromosome Inactive Specific Transcript), which is only expressed on an
inactive X chromosome and is transcribed (or copied) into RNA. Other
probes were used to detect the sex and chromosomal status of each
Ms van den Berg found that while the male embryos showed hardly any
signs of XIST, the female embryos started to show signs of XIST at the
eight-cell stage, and the XIST signal grew stronger at the morula and
“Our results are the first to show that, contrary to what was
previously published, human embryos do inactivate a single X chromosome
before implantation. We have shown that major characteristics that are
present in mouse pre-implantation embryos are present also in human
embryos. This means that dosage compensation is present before
implantation and this could have possible implications for in vitro
culture such as during IVF treatment. Furthermore, our research shows
that X chromosome inactivation in humans is not very different from
other placental mammals, suggesting that it has remained basically
unchanged throughout evolution,” said Ms van den Berg.
“Early failure to perform correct XCI is likely to lead to the demise
of the embryo. A recent publication showed that the sex ratio of
children born after blastocyst transfer in IVF/ICSI treatments is
altered in favour of males. As a result of our finding that XCI occurs
at the pre-implantation stage, further research should be able to
clarify whether culture conditions in the laboratory influence the
growth rate and survival of female embryos and whether this can be
She said that her results also had implications for human embryo stem
(ES) cell research. “Human embryonic stem cell lines are derived from
blastocysts that, we know now, already have, or still have, a form of
XCI. While mouse embryos reactivate the X chromosome in the inner cell
mass at the blastocyst stage so that the derived embryonic stem cells
are completely undifferentiated, it is not yet known whether this occurs
in human embryos. The onset and subsequent steps of XCI in human
pre-implantation embryos occur at a later stage than in mouse embryos.
Thus, it is possible that reactivation of the X chromosome happens also
at a later stage, after the usual time for ES cell derivation. The
current human ES cell lines may, therefore, still have the first wave of
XCI. Indeed, the majority of human ES cells have XCI features.”
This might mean that human embryonic stem cells could behave in a
different and possibly unpredictable way to that expected. If they are
not fully undifferentiated, then they might be unable to transform
completely into the particular tissues that researchers might be trying
to create in the laboratory for therapeutic purposes.
“If the current human ES cell lines are in fact isolated too early,
before X chromosome reactivation happens in the human blastocyst, then
alternative strategies for generating human ES cell lines need to be
developed. Obviously, an important step is to investigate exactly when
and how reactivation of the X chromosome happens in the human embryo and
to use this information in the derivation of new human ES cell lines.
We are planning further research to clarify these issues,” concluded Ms
van den Berg.