Historical Notes
Prenatal diagnosis of fetal genetic
conditions is a standard part of
modern obstetric care. Many of the current methods rely on
invasive methods and are associated with an inherent risk of fetal loss. Consequently there has
been a long term goal for development of NIPD
.
Believe
me. Trust me . The first report of the
occurrence of fetal cells in maternal circulation wasn’t discovered by
Dr Srimanta Pal of Kolkata,
Diagnosis of
presence of foetal cells in maternal
blood dates back to 1893 when the German pathologist
Schmorl identified trophoblast cells in the lungs of women who had died from eclampsia . Till recently however
the existence of fetal cells in
maternal blood was a matter of considerable debate. The main reason for
this dispute was the inability to
successfully and reliably enrich
for these cells which has also hindered their clinical use. In the
meantime this issue has been
addressed and both the fetal
aneuploidies and single gene defects can
now be detected in a non invasive manner using fetal cells enriched from maternal blood . since
the discovery of circulating nucleic
acids in plasma in 1948 many diagnostic applications have emerged .For example diagnostic and prognostic potentials of circulating tumor derived DNA have been demonstrated for many types of cancer. The parallel
development of fetal derived DNA
detection in maternal plasma has
opened up the possibility of NIPT
and monitoring of many
pregnancy associated disorders. In this regard non invasive fetal RhD
blood group genotyping has already been translated into clinical practice. Other applications of circulating DNA in traumatology and transplant
monitoring have also been
reported. The more recent discoveries of circulating tumor derived
ribonucleic acid and fetal
derived RNA have proven
to be equally important as their DNA counterparts Successful prenatal diagnosis of down syndrome by fetal
RNA analysis has recently been reported . However the definite origin and release mechanisms of
circulating nucleic acids have
remained incompletely understood with
cell death being suggested to be associated with such
nucleic acid release.
The history
of prenatal diag of Downs syn : History
& present NRC status of diag of
foetal chromosomal/ genetic diseases. Only small quantities of body fluids it is hoped that they will pave the way for the
development of effective noninvasive approaches Pregnancy
screening for fetal aneuploidy
started in the mid 1960s using
maternal age as the screening test. The research into non invasive and invasive prenatal diagnostic techniques
developed almost in parallel .
On the one hand th need was arising to
ensure the birth of normal progeny
in all cases while on the other hand
it was not possible to eliminate the not insignificant abortion
risks of invasive diagnosis. One
of the first researchers in the non invasive
field was Adinolfi who published
the earliest data in 1974
on the possibility of detecting
three types of fetal cells in the maternal circulation using flow cytometry. Adinolfi suggested the
possibility of using fetal cells present in the maternal circulation
for prenatal diagnosis of chromosome or biochemical
anomalies. Cells are also present in the endocervical canal
where from the 8th
week of pregnancy , it is only possible to obtain trophoblast
cells. This technique has since
been abandoned due to the
scarcity of cellular material available the greater risk
of contamination by cells of maternal available the greater risk of
contamination by cells of maternal
origin and also because the
recovery of the cells is unpredictable despite their potential
use for the early non invasive
diagnosis of sex. Over the
past 40 years much effort
has been spent
on developing NIPD. There has
recently been an upsurge of interest in the analysis of circulating nucleic acids
in blood plasma or serum
as a clinical diagnostic tool. Occasional earlier reports suggested the existence of circulating nucleic acids but the potential clinical implication was not realized until 1996 when DNA with tumor specific characteristics was
demonstrated in the plasma / serum of cancer
patients , this finding
opened up possibilities for non invasive cancer
diagnosis . Potential
applications have been reported in cancer
diagnosis prenatal diagnosis
transplantation and traumatoloty. Some of the findings
are on the verge of being translated into clinical use . DNA
is also now being sought in other body fluids such as urine.
In 1997 the presence
of circulating fetal DNA in maternal
plasma and serum was first
discovered by Lo et al through the detection of Y chromosome specific sequences
in the plasma of women conceived with male
fetuses and has become a useful
tool for prenatal diagnosis in less than
5 years. This discovery has opened
up new possibilities in the development of NIPT
by a source of fetal
genetic material that could be conveniently accessible simply through the collection of a maternal
peripheral blood sample. It has been shown
that cffDNA analysis could
offer highly accurate assessment of fetal genotype and chromosomal make up for some applications. Thus effDNA
analysis has been
incorporated as a part of prenatal screening programs for the management of fetal RhD
blood genotyping to prevent
incompatibility prenatal diagnosis of
sex linked and sex associated
disorders paternally inherited genetic
diseases as well as the prenatal
detection of DS and is a
viable indicator of predisposition to certain obstetric
complications .More recently
there have been significant
new developments with expanding number of
potential applications . Since 1997 in fact
these developments have
been translated into many novel genetic epigenetic
and gene expression markers and are
expected to have fundamental
impact on the future practice
of prenatal diagnosis. Management of pregnancies at risk
of an X linked Menedelian disorder
has changed thanks to the non invasive fetal sex assessment. As
for other mendelian disorders until
recently their study was limited
to those cases paternally
inherited. Nevertheless the
new emerging technologies are also opening the scope to maternally inherited disorders.
Since 1997
many studies have examined the accuracy of prenatal fetal sex determination using effDNA particularly
for pregnancies at risk of an X linked
condition . To evaluate the use of effDNA
for prenatal determination of fetal
sex overall mean
sensitivity was 96.6% and mean
specificity was 98.9% . These results k vary very little
with trimester or week of testing
, indicating that the performance of the test is reliably high. Thus
fetal sex can be determined with a high level
of accuracy by analyzing effDNA . using effDNA in prenatal
diagnosis to replace or complement
existing invasive methods can
remove or reduce the risk
of miscarriage. Further work
should concentrate on the
economic and ethical considerations implementing
an early noninvasive test for fetal sex. The use of circulating
effDNA for the NIPT of fetal chromosomal aneuploidies
is challenging as fetal DNA represents a minor fraction of maternal plasma DNA. In
2007 it was shown that single molecule
counting methods would allow the detection of the presence of a trisomic fetus as long as
masviely parallel sequencing
millions or billions of DNA molecules
can be readily counted . Using MPS
fetal trisomies 21, 13, and 18 have
been detected from maternal
plasma Recently large scale clinical studies have validated the robustness of this approach
for the prenatal detection of
fetal chromosomal aneuploidies. A proof
of concept study has also
shown can be constructed from the maternal plasma DNA
sequencing data. These developments suggest that the analysis of fetal DNA in maternal
plasma would play an
increasingly important role in future obstetrics
practice.
Post
genomics technologies that explore
the proteins and transcripts released
by the placenta into the
maternal circulation offer new opportunities to identify
genes and their protein products
that are key diagnostic markers of disease and might replace
the current screening markers
in use
for prediction of risk of DS. In
the ideal situation these
markers are sufficiently diagnostic
not to require invasive
sampling of fetal genetic
material . Post genomics techniques
might also offer better
opportunities for defining
fetal cell specific markers that might enhance
their isolation from
maternal blood samples. Progresses in
these studies are particularly those
funded by the special noninvasive advances
in fetal and neonatal evaluation
network of excellence . SAFE is a European union
Framework NoE which facilitates the implementation of NIPD for single gene
disorders fetal RhD typing aneuploidy and pregnancy complications. The SAFE project
was set up to implement
routine cost effective NIPD and neonatal
screening through the creation of long term partnerships
within and beyond the European
community and has played a major
role in the standardization of
noninvasive RhD genotyping
. other research using cffDNA
has focused on the amount of
cffDNA present in the maternal
circulation with a view to pre empting various complication of pregnancy . One
if the key areas of interest in the noninvasive
arena is the prenatal detection of aneuploidy pregnancies particularly DS . Owing
to the high maternal DNA background
detecting of effDNA from
maternal plasma is very difficult . consequently research in this area is now more focused on effDNA to produce
new biomarkers.
Proteomics
based identification of biomarkers
for fetal abnormalities in maternal
plasma amniotic fluid and
reproductive fluids has made significant
progress in the past 10 years. This is attributed
mainly to advances in various
technology platforms associated
with mass spectrometry based techniques. As these techniques
are highly sensitive and require
only small quantities of body
fluids it is hoped that they
will pave the way for the development
of effective noninvasive
approaches without subjecting the developing fetus to the same degree of harm
as current invasive procedures . It
is possible that these developments will include same day analyses thereby permitting rapid intervention when necessary. To date a host of body
fluids such as maternal serum and
plasma amniotic fluid
cervical fluid vaginal
fluid urine saliva or fetal material such as placental trophoblast fetal membranes or cord blood have been used
successfully in the quest to develop markers for a number of pregnancy rerated
pathologies. The emergence of
proteomics has come as a major platform
technology in studying various types of fetal
conditions and developing markers for pregnancy related disorders such fetal aneuploidy preterm birth
pre eclampsia inter amniotic infection and fetal stress. Comparisons’ of proteomes of normal
fluids with those from aneuploidy pregnancies have revealed a host of candidate
markers that still need to be
verified. In parallel with proteomics
there is
interest in other emerging
techniques such as RNA single
nucleotide polymorphism analysis
or quantization of fetal DNA by shotgun
sequencing . Although these genomic techniques hold much promise discovery of additional markers
via quantitative proteomic
comparisons could drastically improve current conventional screening at reasonable cost Proteomics based biomarker discovery
is applicable to detection of not
just aneuploidies but also other pregnancy related diseases. Should the development of these markers be
successful then it is to be envisaged
that proteomic approaches will become
standard fo care for a number of
disease conditions associated with feto maternal health.
Prenatal
Screening and Diagnosis of Down Syndrome
Down
syndrome occurs when a person has three
copies of chromosome 21 rather than
two . It is the commonest
congenital cause of mental retardation. Noninvasive screening based on biochemical analysis of
maternal serum or urine or fetal
ultrasound measurements, allows estimates
of the risk of a pregnancy being affected and
provides information to guide decision about definitive testing. Two thirds of the patients with DS
are born by women below 35 years of age.
An extension of invasive
postnatal diagnosis to these women is problematical due to the operative risks and the relatively high costs .
Therefore noninvasive screening
methods are sought apt to
identify groups at high risk such as biochemical screening methods
applied at 16 weeks of pregnancy the
first trimester tests and ultrasound screening at 10 to 12 weeks . A large fluid cushion over most of the back was documented not only in most cases
of trisomy 21 but in trisomies 18 and 13 and in Turner syndrome
as well . Only few chromosomally
normal fetuses with the same
peculiarity were observed . Systematic
first trimester screening for nuchal fluid accumulation seems to be a recommendable method for the detection of chromosome
anomalies. It compares favorably with
methods of MSS performed at 16 to 18
weeks which require a manyfold higher number of invasive procedures . Prenatal diagnosis
of trisomy 21 currently relies
on assessment of risk followed by
invasive testing in 5% of pregnancies at the highest estimated risk . Selection of the high
risk group by a combination of maternal
age and second trimester
maternal serum biochemistry
gives a detection rate of about
60% Assessment of risk by a combination of maternal age and fetal nuchal translucency thickness
measured by ultrasonography at 10 to 14
weeks of gestation has been investigated Selection of the high risk group for
invasive testing by this method
allows the detection of about 80 % of
affected pregnancies.
However even this method of risk assessment
requires about 30 invasive
tests for identification of one affected fetus.
Studies evaluating tests of maternal serum in women at 14 to 24 weeks of gestation for DS compared with a reference standard either chromosomal verification or macroscopic postnatal
inspection have been made.
Data were extracted as test
positive / test negative results
for Down’s and non Down’s pregnancies allowing estimation of detection rates and false positive rates . Twelve different tests
have been matched with
maternal age alpha fetoprotein
unconjugated estriol total human
chorionic gonadotropin free B- hCG
free a-hCG inhibin a specificity protein 2. Cancedr antigen 125 or
carbohydrate antigen `125 tropinin
pregnancy associated plasma
protein A placental growth
factor and proforma of eosinophils major basic protein Meta analysis of 12 best
performing or frequently
evaluated test combinations showed double and triple tests significantly outperform individual markers detecting 6 to 7 out of every 10 DS pregnancies at a 5% false positive rate. Tests
additionally involving inhibin
performed best but were not
shown to be significantly better than
standard triple tests in direct comparisons. Significantly lower sensitivity occurred in women over the age of 35 years. Women who miscarried in the over 35 group were more likely to have been offered an invasive test to verify a negative screening
results whereas those under 35 were usually
not offered invasive testing for
a negative screening result . Pregnancy loss in women under 35 therefore
leads to under ascertainment of
screening results potentially missing a proportion of affected pregnancies and affecting the accuracy of the sensitivity .
Tests involving two or more markers in combination with maternal age are significantly more sensitive than those involving one marker . The value of
combining four or more tests or including inhibin have not been
proven to show statistically
significant improvement Further
study is required to investigate
reduced test performance in women aged over 35 and the impact
of differential pregnancy loss on
study findings.
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