Since the birth of Louise Brown in 1978, more than a million babies have
been delivered worldwide with assisted reproductive technologies or ARTs. An
estimated one in ten people of reproductive age are infertile in the
industrialized countries, and ARTs are now involved in 1% to 3% of annual
ARTs include in vitro fertilization (IVF), induced ovulation
(IV) and intracytoplasmic sperm injection (ICSI), which, in turn, form the
basis of all newer reproductive and related technologies such as cloning,
somatic cell nuclear transplant, genetic modification of germ cells, and
recently, transformation of stem cells into germ cells.
Simultaneously as the new reproductive technologies are enthusiastically
developed and exploited by biotech companies, however, evidence is emerging
that ARTs themselves carry risks to the unborn (see Box 1).
Birth defects associated with ARTs identified between 2002 and
A study in the United States reported a 7.3-fold relative
increase in incidence of an extremely rare urogenital system birth defect
during the first trimester of gestation.
IVF was reported to increase risk of retinoblastoma (malignant
tumour of the retina) in the Netherlands 4.9 to 7.2 fold compared to non-IVF
A study funded by the US National Institutes of Health found
three (5%) out of 65 children with Beckwith-Wiedemann syndrome - characterized
by enlarged tongue and predisposition to rare cancers - were conceived via IVF.
Overall, only 0.8% of births in the US occur after IVF.
In Brazil, four children aged from 6 months to 3 years with
several types of cancer were born and diagnosed between 1996 and 2000, whose
IVF took place in Sao Paulo. These are out of approx 2000 live births during
the five-year period as the result of IVF. The annual incidence rate for cancer
for children aged 0-4 years in Brazil was 117.5 cases per 1 000 000. Thus, only
one case of cancer would be expected among the IVF children.
A large study in Sweden compared the development of
neurological problems in 5680 children born after IVF with 11 360 matched
controls. Researchers found that children born after IVF are more likely to
need habilitation services than controls, the overall risk (OR) was 1.7. For
singletons (single births), the relative risk was 1.4. The most common
neurological diagnosis was cerebral palsy, with increase overall risk of 3.7
after IVF, and 2.8 for IVF singletons. Suspected developmental delay was
increased four-fold in children born after IVF.
In Western Australia, the rates of major congenital birth
defects in children conceived by IVF, ICSI and control cohorts were found to be
8.6%, 9% and 4.2% respectively.
In Northern Finland, 304 IVF children born in 1990-1995 were
compared with 569 randomly chosen matched controls. The overall IVF risks for
incidences of preterm birth was 5.6, very low birth weight, 6.2, low birth
weight, 9.8, neonatal morbidity, 2.4, hospitalization, 3.2. The prevalence of
heart malformations was four-fold in the IVF compared to controls. With the
exception of heart malformations, most of the risks were attributed to multiple
births after IVF.
Multiple pregnancy emerged as the most important, albeit not the only
risk factor. The European Society of Human reproduction and Embryology (ESHRE)
reviewed its guidelines on good practices in IVF laboratories in 2002, and
recommended aiming for singleton pregnancies. It is claimed that methods of
single embryo transfer have been refined to result in acceptable pregnancy
rates, made feasible by improved in vitro techniques in identifying good
However, a review in the Lancet points to in vitro germ
cell and embryo culture techniques as the cause of certain birth defects.
Three studies in 2003 all report an unexpectedly high incidence of
Beckwith Wiedemann syndrome (BWS) in children conceived with ARTs. Patients
with BWS have abnormalities at chromosome 11p15 associated with organ
overgrowth and abdominal wall defects as well as increased risk of embryonal
tumours. Six of 149 cases were reported from a British BWS registry, the same
numbers in a French registry and a further 7 in the USA. These frequencies are
extraordinarily high for such a rare congenital condition, representing a
significant 4.2-fold increase associated with ARTs.
These findings are reminiscent of reports of sporadic cases of the
genetic imprinting disorder, Angelman Syndrome, which has also been
linked with ARTs. Angelman Syndrome is characterized by severe mental
retardation, motor defects, lack of speech and a happy disposition, and is
linked with a loss of function of the maternal allele (copy of gene inherited
from the mother) of UBE3A on chromsome 15.
About 50 genes are differentially expressed according to their origin in
either the oocyte (egg cell) or spermatozoon (sperm cell). These
imprinted genes have roles in growth and development and in tumour
suppression (for example, retinoblastoma has been reported by Dutch
investigators to be more frequent among ARTs children than normal). At the
imprinted genes, only one allele is active (maternal or paternal) and the
inactive allele is epigenetically (developmentally) marked, by chemical
modification of the histone protein bound to DNA, or by adding a methyl group
(-CH3) to the base cytosine on the DNA, or both.
Early in development of the fetal germ cells in both sexes, the
germ-cell genomes are erased of methylation marks on the imprinted genes.
During maturation of sperm and egg cell, however, re-methylation of the
imprinting alleles takes place. DNA methylation, almost invariably associated
with repression of transcription, targets one of the two parental alleles to
silence it. After fertilization, there are further changes in overall genomic
methylation in specific cellular lineages of the embryo but importantly,
imprinted alleles are protected from these waves of demethylation and
remethylation to maintain their proper dosage effects.
Thus, major epigenetic events take place during both germ-cell
development and pre-implantation stages when ART procedures are performed,
possibly interfering with the proper establishment (in gamete culture) and
maintenance (in embryo culture) of genomic imprints.
Both BWS and AS are associated with imprinted gene clusters. In about
50 to 60% of sporadic cases of BWS, and 5 10% of the cases of AS, an
epigenetic defect is involved rather than a mutation in the gene. In contrast,
all cases of AS and 13 of 19 cases of BWS linked to ARTs were due to epigenetic
defects, involving loss of methylation in the maternal allele.
Gene expression - and methylation status - in animal embryos is known
to be affected by culture conditions. Notable changes in expression of other
genes take place in embryos in culture, but effects on imprinted genes are
different in character and are unlikely to be reversible adaptations to altered
environmental conditions. In some farm animals, embryo culture and cloning
technologies carry high risks of neonatal overgrowth, morbidity and mortality,
which, in sheep are associated with loss of imprinting at the Igf2r locus. The
so-called large offspring syndrome in animals is reminiscent of BWS in man.
Some researchers have made connections between the effects of the
manipulation of gametes and embryos in IVF and those resulting from the
manipulation of the maternal diet, even briefly, during early pregnancy. Diets
both high and low in protein content can have detrimental effects on embryonic
and neo-natal development. High protein diets in sheep during the
peri-conceptual period have been linked with low embryo survival and high birth
weights similar to the large offspring syndrome. Low protein diets during early
pregnancy in rats were found to significantly reduce birth weight of pups.
Experimental studies in animal models have clearly established links between
poor maternal nutrition to altered prenatal growth and adverse outcomes in
terms of cardiovascular and metabolic function in adult offspring.
Intriguingly, similar results following embryo culture are now reported:
lighter fetal weight in mice and increased post-natal adiposity, together with
body weight gain and abnormal organs in adults. Compared with in vivo
derived embryos, culture reduced total cell number and inner cell mass (which
eventually gives rise to the fetus). Adding granulocyte-macrophage colony
stimulating factor appeared to mostly to overcome those effects.
In vitro culture in ruminants is linked with defective placenta
formation. In cow, in vitro embryo production can yield fetuses with
abnormal allantoic development and failed blood vessel supply to the developing
placenta at day 35 of pregnancy. Similar effects were observed in sheep. But
oversized fetuses or placentas have also been reported in cows.
Epigenetic modification in ART embryos is thus a component of a broader
causal model linking environmental stress factors with disturbances to
development though both transcriptional and epigenetic modification of gene
expression (see "Diet trumping
genes", this series).
In yet another setback to ARTs, scientists at Brown University,
Providence, Rhode Island in the United States reported in October 2003 that
using frozen embryos in fertility treatments raises the risk of ectopic
pregnancy 17 times. They compared 2452 cycles of IVF using fresh embryos with
392 using frozen transfers. Ectopic pregnancies were 1.8% in the former group
and 31.8% in the latter.
Ectopic pregnancies are potentially fatal to the embryo, which gets
stuck in the fallopian tube. It causes agonizing pain and can be fatal for the
woman if not detected early enough and the embryo removed.
When a woman first has IVF, one to three fresh embryos are usually
transferred into the uterus. Within the past decade, as increasing numbers of
couples are choosing to freeze some of the spare embryos, giving them a chance
to try again if the first attempt fails, thus avoiding the painful process of
hormone treatment to induce ovulation.
Storage of frozen embryos also allows patients who have to undergo
chemotherapy treatment and other women to create fertilized embryos and delay
Dr. David Keefe, the lead researcher, expressed surprise at the finding,
"We did not expect it to be so high and we obviously need more research." He
said. He thought it could be the thawing process that may disrupt the
development of the embryo, making it more prone to stick to the fallopian tube.
A total of 250 000 babies have been born through frozen embryo