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AT THE Hammersmith Hospital, my colleagues and I have succeeded in distinguishing
male from female embryos at the earliest stages of development. The tests
that we have now developed are described in Nature this week. They offer
couples who are at risk of transmitting certain genetic diseases that affect
only boys the prospect of starting normal pregnancies, secure in the knowledge
that they will give birth to unaffected girls.

Earlier experiments on animal embryos and spare human embryos donated
for research showed that we could remove a cell from an eight-cell embryo
and determine its sex without damaging the rest of the embryo. Further studies
on human embryos donated for research may soon enable us to offer more sophisticated
tests to couples at risk of passing on other severe genetic diseases such
as cystic fibrosis, beta thalassaemia and Duchenne muscular dystrophy.

To these people, the prospect of ‘preimplantation diagnosis’ – the detection
of genetic disease in very young embryos in the laboratory, before implantation
and pregnancy has begun – may seem a godsend. First seriously proposed some
five years ago, preimplantation diagnosis would enable only those embryos
that were free of the genetic disease to be transferred to the womb. The
woman could begin a pregnancy in the knowledge that it was unaffected (see
‘Embryo research and genetic disease’, by Marilyn Monk, New ÐÓ°ÉÔ­´´, 6
January 1990).

We have now established that it is feasible to carry out a form of preimplantation
diagnosis in the special case of the so-called X-linked genetic diseases
which affect only boys . To do this, we have devised tests that can distinguish
female embryos from male ones. Although this approach does not specifically
‘diagnose’ the presence or absence of the defective gene, the process involved
in doing so would be similar to the one we have carried out.

We based our approach on the techniques of in vitro fertilisation (IVF)
that are now well established for the treatment of infertility. In this
procedure, women take hormonal drugs that cause their ovaries to produce
several mature eggs in a single cycle. These eggs are then collected and
fertilised in the laboratory. If the clinic has several embryos to work
with, there is an increased chance of identifying unaffected or female embryos.

The day after the eggs are inseminated in the laboratory, we check them
to see if fertilisation has been successful. Normally, fertilised embryos
are then cultured for a further two days, by which point most will have
reached the eight-cell stage. We have now shown that we can determine the
sex of these embryos, by removing one of their cells for testing, without
damaging them.

To perform the test, or biopsy, we first had to cut a hole in the glycoprotein
coat, known as the zona pellucida, that surrounds the embryo. We did this
by placing a micropipette close to the embryo and releasing a stream of
an acidic medium onto the zona. We then introduce a second, larger pipette
through this hole and remove one or two cells by suction. While the biopsied
embryo is returned to culture, we prepare the single cell that we have removed
from it for analysis.

The task is now to determine the sex of this single cell: that is, does
it contain a Y chromosome or not? We chose the fairly new technique known
as the polymerase chain reaction, which ‘amplifies’ DNA (see ‘Genes unlimited’,
New ÐÓ°ÉÔ­´´, 14 April). The technique enables us to make thousands of
copies of a particular DNA sequence – in this case, one found on the Y chromosome.
This sequence is easy to identify with a specific DNA probe, because it
is already repeated many times on the Y chromosome.

In this way, we can quickly produce enough DNA to detect the sequence
using the standard tools of molecular biology. The whole procedure is so
rapid that we can biopsy a group of embryos in the morning, amplify the
DNA and identify those that are male, and transfer selected female embryos
to their mother in the evening.

But we could not have used this procedure without being sure it was
safe. Before transferring biopsied embryos to a woman’s uterus, we had to
find out whether the biopsy affected their ability to develop to blastocysts
– balls of a few hundred cells. So we first compared the development of
biopsied and intact embryos in culture.

The results showed that the biopsy had not reduced the proportion of
embryos that reached the blastocyst stage. Indeed, a much higher proportion
‘hatched’ from the zona pellucida than did the intact embryos. This hatching
must happen if the blastocyst is to attach itself to the lining of the uterus
and become implanted in it. In culture, the zona pellucida can become hardened.
Some researchers have recently suggested that this hardening of the zona
might prevent some embryos from hatching and so contribute to the low rate
of implantation after IVF. Perhaps, then, the ‘drilling’ of the zona to
biopsy embryos is beneficial in itself, increasing the chances that an embryo
will implant.

To assess the viability of biopsied embryos further, Kate Hardy in this
laboratory used a technique that distinguishes between two types of cell
in the blastocyst. One group of cells, called the trophectoderm, goes on
to form the placenta, while the other cells, called the inner cell mass,
eventually develop into the fetus. Using different combinations of DNA-specific
fluorescent dyes, Hardy was able to stain the trophectoderm cells orange,
and the inner mass cells green, and count them under the microscope.

She found that biopsied embryos reaching the blastocyst stage did have
fewer cells than intact embryos, but only in proportion to the reduction
in the cellular mass after biopsy. So, for example, the eight-cell embryos
from which a single cell had been removed had about seven-eighths the total
number of cells of intact embryos. Most importantly, the number of cells
in the inner cell mass was not disproportionately reduced.

Finally, we checked the health of biopsied embryos in another way, by
working with Karen Martin and Mike Hooper in Henry Leese’s laboratory at
the University of York. We used a noninvasive measure – one that involves
no physical interference with the embryos – which Leese has developed over
the past 10 years. He has devised a way of measuring the uptake of the energy-rich
molecules pyruvate and glucose. This is done by culturing the embryos in
small droplets of medium with known concentrations of these chemicals, under
a layer of oil to prevent evaporation.

After 24 hours, we remove the embryos and transfer them to fresh drops,
then repeat the process. Meanwhile, the spent medium from the original droplet
is mixed with an enzyme cocktail which causes another chemical to fluoresce,
depending on the amount of substrate present. Using this method, we can
measure chemicals in the small volumes of medium used to incubate the embryos.

We found that the biopsied ones took up less pyruvate and glucose than
intact ones, but, again, only in proportion to their reduced cellular mass.
So apart from a reduced number of cells, the development of human embryos
up to the time they implant was unaffected by the removal of one or two
cells at the eight-cell stage.

Last September, we obtained approval for a clinical trial of embryo
biopsy from the research ethics committee of Hammersmith Hospital and from
the Interim Licensing Authority, set up by the government in 1985 to oversee
research on human embryos.

Over a period of six months, we have treated five couples who are at
risk of transmitting X-linked diseases. These disease include X-linked mental
retardation, adrenoleukodystrophy, Lesch-Nyhan syndrome and Duchenne muscular
dystrophy. Each of the five couples had in the past had affected pregnancies
terminated after undergoing conventional diagnosis. They had found these
experiences very traumatic, and they now chose to have IVF and the transfer
of female embryos.

In each treatment cycle, we biopsied an average of five embryos and
were usually able to identify several female embryos and to transfer two
to the uterus. We consider this to be the maximum it is safe to transfer,
to reduce the risk of multiple pregnancies.

Several of the women soon showed biochemical signs of being pregnant.
We could detect in their bloodstream a hormone known as human chorionic
gonadotrophin (hCGn). Embryos release this chemical only after they have
become implanted in the uterus and begun to develop. In two of these cases,
ultrasound scans have confirmed that the women were pregnant. After four
weeks, it was clear that twins were developing in both cases.

To confirm the sex of the twin fetuses in the two established pregnancies,
and so the accuracy of our tests, Charles Rodeck, a gynaecologist at Queen
Charlotte’s Hospital, performed the prenatal test known as chorionic villus
sampling, at 10 weeks of pregnancy. This procedure involves removing a bit
of tissue from the placenta, so that the fetal DNA can be analysed. All
four fetuses are female and have normal complements of chromosomes. These
early results are encouraging. Two out of the five women treated over the
past six months are now pregnant. Provided that both sets of twins are not
identical – that is, they have resulted from the implantation of both of
the transferred embryos rather than one that divided – four out of 17 biopsied
embryos that were transferred have developed successfully.

But it is too early to forecast success rates for pregnancy after preimplantation
diagnosis. Although one of the two women became pregnant after only one
treatment cycle, the other had two treatments, and many women may need more
that this before the procedure works. Despite these uncertainties, many
couples may prefer this approach to prenatal diagnosis, which may involve
several abortions over a number of years.

The House of Commons will vote this week on whether to ban research
on human embryos altogether or allow strictly regulated research on embryos
up to 14 days. Our new-found ability to identify the sex of early embryos
to prevent X-linked diseases from being passed on is a good example of the
need for regulation. Many families with several children all of the same
sex would like to complete their families with a child of the opposite sex.
But we do not think it is ethical to consider choosing a baby’s sex using
the techniques we have developed.

At the moment there is nothing to stop other clinics offering just such
a service for choosing a baby’s sex. But a statutory authority, to be created
by the Embryo bill now before parliament, would allow couples at risk of
passing on a genetic disease to go ahead with the implantation of an embryo
of known sex, while prohibiting this simply for choice.

Yet there is a long way to go before preimplantation diagnosis could
be offered routinely to couples at risk. The technical challenge is to perfect
reliable ways of detecting just one or two copies of a defective gene in
a single embryonic cell. (Remember that the sexing of embryos is now possible
only because it relies on the detection of DNA sequences repeated hundreds
of times on the Y chromosome.) Research on spare human eggs and embryos
is essential if we are to establish any of these techniques in clinical
practice. We hope that MPs will vote to give us the opportunity of making
every effort to ensure that safe and reliable biopsy and diagnostic tests
are developed.

* * *

FOR BOYS ONLY: THE TRAGEDY OF X-LINKED DISEASES

THE Victorians first recognised a class of inherited diseases that affect
only boys. These conditions are passed to successive generations through
unaffected women ‘carriers’. Queen Victoria herself was a carrier of one
such disease, haemophilia.

Only boys are affected because the faulty gene happens to lie on the
X chromosome, one of the so-called sex chromosomes. Girls inherit two X
chromosomes, one from each parent. With one normal X chromosome, they are
shielded from the disease. Boys, on the other hand, with one X chromosome
from their mother and one Y chromosome from their father, will suffer from
the disease if they happen to inherit their mother’s faulty X chromosome.
More than 200 X-linked conditions have been identified. They vary in severity
from mild forms of anaemia, haemophilia and X-linked mental retardation
to fatal diseases such as adrenoleuokodystrophy, Lesch-Nyhan syndrome and
Duchenne muscular dystrophy.

The molecular basis of many of these diseases is now known and prenatal
diagnosis is possible. So women who find that they are carrying an affected
fetus have the option of an abortion. But if the molecular defect has not
been identified, the only alternative is to determine the sex of the fetus,
with the option of aborting the males. This approach cannot distinguish
normal males from abnormal ones, or tell the difference between ‘carrier’
and healthy females.

Dr Alan Handyside is a senior lecturer in the Institute of Obstetrics
and Gynaecology of the Royal Postgraduate Medical School at the Hammersmith
Hospital in London.