Introduction abnormalities by using carrier tests. Innovative technologies



has been more than 150 years since scientists discovered cells,
chromosomes, and genes. In 1859, the genetical theory of
natural selection had been proposed by Charles Darwin5. Six
years later, Gregor Mendel identified
inheritance pattern from his
pea plants, resulting in Mendelian principles7. Then the world
of genetics has been explored continuously. One hundred years later, in
1959 Jerome Lejeune and his colleague found that an additional
chromosome, trisomy 21, is the cause of Down Syndrome, after the discovery
of karyotyping techniques. Later on, the knowledge regarding the
genetic disorders was dramatically increased alongside the development of advanced

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our knowledge, genetic diseases are grouped into two mains categories. First is
aneuploidy, an abnormal number of chromosomes either an extra- or absent one.

The other major category is inherited genetic diseases, mostly resulting from
mutation. Conceiving a child affected with genetic disorders would lead to
disability, morbidity or even death, and reduce their quality of life, as well
as consume a number of resources in order to take care of that child. Therefore,
it is necessary to early identify parents’ possibility of having an affected offspring.

the genetic screening test has been established in order to detect fetuses with
aneuploidy, and to identify whether a couple have risk of conceiving an
offspring with genetic abnormalities by using carrier tests. Innovative technologies
have been developed and applied to improve the prenatal genetic tests from
traditional karyotyping in the 20th century to recent discovery of whole
exome sequencing (WES) and whole genome sequencing (WGS). Each advanced method
has both benefits and drawbacks which need to be considered and discussed in
term of the application, cost-effectiveness as well as the ethical issues. Because
we do not only detect the genetic diseases or probability of having genetic disorders
in the offspring, but also provide parents an opportunity to terminate a
pregnancy.  The issues regarding the
development of genetic screening tests and it’s both advantages and
disadvantages including future directions of these tests will be discussed in
this essay.




our rapidly increasing understanding of the genetic mechanism, prenatal
diagnostic methods for different genetic diseases have been
developed. The establishment of amniotic fluid cell
culture for detecting fetal chromosomal
disorders, in 1966, and the change in the law
and people’s perception of abortion in pregnant women
at high risk of fetal chromosomal abnormality would have been the beginning of
the history of prenatal screening and diagnosis8. 

In the 1980s, the aim
of prenatal diagnosis was mainly to identify trisomy 21 (Down
syndrome), however, another form of chromosome abnormalities and
rare diseases could also be identified accidentally with the

the 20th century, there were two well-known procedures,
used generally for prenatal diagnosis; amniocentesis and chorionic
villus sampling (CVS)9. Although both
could prenatally detect chromosome abnormalities and single gene disorders, they were also considered as
invasive procedures which carry the risk of fetal loss. 




previously stated, amniocentesis had been introduced
and developed as a diagnostic procedure for high-risk patients
in the 1970s. It is normally performed at the gestational age of 16-18
weeks9-12. Some institutions offered this procedure at an early
stage of pregnancy (

The integral molecular tests, including chromosomal microarray
analysis (CMA), interphase fluorescent in situ hybridization (I-FISH),
quantitative fluorescence polymerase chain reaction (QF-PCR),
and comparative genomic hybridization (CGH), provide an opportunity to identify the
wider range of chromosomal abnormality, for example, aneuploidy, translocation, 

mosaicism, and pseudo-mosaicism.  

the advantages, there are some disadvantages of
performing the mid-trimester amniocentesis. Firstly, it would lead to maternal and fetal complications9.

Bacterial invasion occurring through puncture sites, skin or even bowel,
possibly cause maternal infection, septic shock or death in a very severe case.

Moreover, the needle insertion would result in various degrees of fetal injury from
a small skin dimple to severe internal organ injuries. Secondly, it might
increase the risk of preterm labor as well as other third-trimester complications,
including chorioamnionitis, oligohydramnios, and preterm premature rupture of
the newborn, as a consequence of unnoticeable amniotic fluid leakage. Thirdly, errors could occur by
maternal cell contamination, laboratory error, and typographic mistakes13.

Previously, the error rates of amniocentesis were reported about 0.1-0.6% in
the 1970s14. These were significantly reduced to 0.01-0.02% in the
1990s15, 16, possibly stemming from more developed laboratories and
more experienced operator. Nonetheless, following the introduction of prenatal
cytogenetic diagnosis, some chromosome abnormalities, such as low-level
chromosome mosaicism, uniparental disomy, subtle structural abnormality, and
microdeletion syndrome, can be undetectable. Moreover, the relatively late diagnosis
during mid-trimester pregnancy would lead to tight mother-child bonding and ethical
issues regarding the termination of an abnormal fetus. Consequently, decision
making after the diagnosis might become more problematic. Although there is an
improvement in ultrasonographic technology which could support early amniocentesis
in the first-trimester, its benefits must be balanced against the higher risk of pregnancy losses and other complications such as amniotic


villus sampling

In the late 1980s, chorionic villus sampling (CVS) had
been established and become more popular than amniocentesis17.

The reason why physicians shifted their practices towards CVS is that it can be
done in the first trimester (10-14 weeks gestational age). In addition, it is
also safe, effective and reliable for the diagnosis of chromosomal, molecular
and biochemical disorders, comparable to mid-trimester amniocentesis. Chorionic
villus, which is floating freely within the intervillous space, will be
obtained by inserting a needle through the maternal abdomen or introducing a
catheter into the cervical canal.

Confirmation of the sampling tissue is necessary to
ensure that there is the presence of required placental tissue and the amount is adequate enough to
reduce maternal cell contamination (10 mg of well-formed villus tissue)17.

CVS samples will be analysed by tissue culture together with direct analysis,
which the latter method could provide results within two days. Despite that
advanced cytogenetic analysis applying through CVS sample is quite similar to
amniocentesis, the advantages of this method are still noticeable. First, it
expands the opportunities to detect genetic disorders, including mosaicism, at
an early stage of pregnancy, especially in the families at risk for single gene
disorders17. Additionally, CVS may require a much shorter time
period for the prenatal cytogenetic results in the setting with technical
feasibility. Concerning the perinatal and maternal morbidity, there is the less
reported rate of premature birth, premature rupture of membrane and maternal morbidity
in this procedure, as compared to amniocentesis. Hence, this procedure has a
significant advantage in term of leading to less maternal mental distress.

Nevertheless, there are some possible drawbacks of CVS which
need to be discussed with the families. The most concerned one is that it may be
related to limb reduction defects in fetuses, which was reported with an event
rate of 1-7.3:10000 cases in the previous studies9, 17-22.

One pathogenesis which could explain this complication is that excessive
placental tissue sampling, usually found in the less experienced centres, can
result in massive vascular disruption of the placenta. Subsequently, profuse
fetal hemorrhage, hypovolemia, and hypoxia may cause the thin-walled peripheral
vascular rupture, tissue necrosis and subsequently limb reduction9.

Another concern is about maternal infection which is
frequently detected in the patients who underwent the transcervical CVS. The
explanation of bacterial invasion in this method is the same as in amniocentesis
which was mentioned earlier in this essay. However, the occurrence of post-CVS
chorioamnionitis is very low, causing miscarriage in only 0.3%17. Unlike
the transcervical CVS and amniocentesis, the transabdominal-CVS may result in
less number of patients with chorioamnionitis or amniotic fluid leakage because
the needle does not break the amniotic membrane9. Interestingly,
another study41 reported the rate of maternal bacteremia post
transabdominal CVS is higher than post transabdominal CVS.

Moreover, the most important issues which need to be pointed
out are about the false-positive and false-negative results of this test and
the pregnancy loss rate. Even though direct visualization of the chorionic
villi can provide a very fast outcome, it also comes up with occasionally
inaccurate results, depending on different laboratories. Regarding the rate of
miscarriage, some studies reported that spontaneous abortion post-CVS is higher
than that for amniocentesis, however, the rate is different among different
centres, based on operators’ level of experience10. Lately, there is
a conclusion that the loss rates for both amniocentesis and CVS are similarly
at 0.1% to 0.3% when performed by skilled operators3. Hence, this
information should be discussed with the family before both physicians and
families decide to choose a suitable procedure for prenatal diagnosis.

Non-invasive screening tests

The previous section has shown that both amniocentesis
and CVS are highly invasive, hence, a number of non-invasive screening test
have been developed. Primarily, the aim of screening tests is
mainly for the early detection of Down syndrome and other types of aneuploidy. Thus,
multiple maternal serum markers such as maternal serum human chorionic
gonadotrophin (hCG), the free-? subunit of hCG, pregnancy-associated plasma
protein (PAPP)-A, unconjugated estriol (uE3), inhibin A and Alpha-fetoprotein
(AFP), and ultrasound nuchal translucency (NT) were initiated and integrated in
the prenatal screening protocol. Different serum markers have their own screening
performance, which is dependent on the different time period of gestational age25.

The best marker during the first and second trimester are PAPP-A and free ?-hCG,
respectively. Whilst, AFP and uE3 are the weakest ones. Despite its
own performance in screening Down syndrome, its cutoff levels are diverse among
different institutions. Therefore, a statistical model was applied to develop a
suitable protocol for the use of combined markers.

The “Combined” test, including NT, PAPP-A and
either hCG or free ?-hCG, is proposed to use for the first-trimester screening.

Whilst, the “Quad” test, using hCG or free ?-hCG, AFP, uE3 and inhibin A, is the
best test for the second-trimester. However, the more complex the protocol is,
the better performance will be acquired. Hence, “Contingent” method is applied
by utilising both first- and second-trimester tests to identify 1% of the
highest risk pregnancy during the first-trimester in order to offer a prenatal
diagnostic test. Blood test for Quad markers will be taken from the other
15-20% of women who have the lower risk of pregnancy in order to re-evaluate
their risk during mid- trimester25. Nevertheless, all these markers have
been used for screening the possibility of having fetal aneuploidies, which
need to be confirmed by the diagnostic tests afterward.

Subsequently, the advanced ultrasonography is
added to the screening protocol both in first- and second-trimester to improve the
screening yield. The additional ultrasonographic findings in the early
pregnancy included absent fetal nasal bone, increased frontal-maxillary facial
angle, tricuspid regurgitation detected by Doppler ultrasound, and the absent
or reversed flow of ductus venosus. The markers for the mid-pregnancy are femur
or humerus length, and the “facial profile” markers including nuchal skin-fold,
pre-nasal thickness, and nasal bone length25. Nonetheless, the
operator-dependent nature of this test is an arguable problem, the performance
may increase only in the sufficient skill hands.

Non-invasive prenatal testing (NIPT)

Turning now to the recently
discovered DNA screening technology, cell-free fetal DNA (cffDNA) analysis, which
was first disclosed by Lo YM, et al in
199726. Following that, we found approximately 10% of cell-free DNA (cfDNA)
in the maternal circulation are basically fetal or placental fraction, which
could represent the fetal genome (Figure 1). By quantifying the proportion of
chromosome from DNA fragments, using specific tags from the chromosome of
interest, or implementation of cytogenetic analysis we can identify a much wider
range of chromosomal abnormalities than the traditional karyotyping method,
including; aneuploidies (trisomy 13, 18, and 21), sex chromosome aneuploidies (Turner
syndromes), other autosomal aneuploidies, and also microdeletion3, 25, 27.

Additionally, the cffDNA could be detectable from 4 weeks of gestation and its
proportion will increase with gestation, hence, this test can easily be done at
an early stage of pregnancy, recommended after 10 weeks of gestational age, without
any risk of miscarriage.

Figure 1. Cell-free fetal DNA analysis is considered as a
non-invasive method for prenatal genetic testing compared to amniocentesis and
chorionic villus sampling28

In terms of accuracy of the test, studies in
the women at increased risk of fetal aneuploidy reported sensitivity and false
positive rate for Down syndrome are 99.4% and 0.16%, respectively. Similar to
the rates in trisomy 18 which are 96.6% and 0.05%, whilst those in trisomy 13
are 86.4% and 0.09%, respectively27. It is noticeable that the false
positive rate of cffDNA testing is very low, so this test can reduce the
requirement for the diagnostic testing. Previous studies showed that the cause
of the error is probably caused by placental and maternal mosaicism. Based on
our knowledge, the positive predictive value is conditional on the prevalence
in that population, thus, the detection rate of this test may be lower in the
lower risk population. However, this test still has a considerably higher rate
of detection than those of the serum marker screening protocol.

Despite the usefulness of this non-invasive
and highly sensitive screening test, there are some limitations. Importantly,
the percentage of fetal fragment circulating in maternal blood should be taken
into account before interpretation of the results. Laboratories should report
“fail to report a result” or no-call result in a low proportion of fetal cfDNA

paper NHS ***

Additionally, there is an argument concerning
the screening performance of this test which is not sufficient enough to
replace the diagnostic tests. Some centres implementing cffDNA analysis may consider
to omit the first trimester ultrasonography or biochemical screening, however, they
should re-consider that cffDNA screening may fail to identify some chromosomal
abnormalities that are detectable by another diagnostic methods25, 27.

Another limitation is regarding its high cost25
which may prohibit the applicability of this test in the public health system.

Consequently, it would not be an available screening test for every woman,
especially in the ones in low socioeconomic countries. This would have an
effect on the families both psychologically and economically. Moreover, we
still need further studies regarding the implication of advanced technologies to
analyse fetal genetic materials more precisely and cost-effectively, especially
in the setting of low fetal fraction.

carrier screening and preimplantation genetic diagnosis

As was pointed out in the introduction to
this paper, the other type of genetic screening test is carrier tests to
identify the parents’ risk of having children with specific autosomal recessive
disorders. Previously, there was an ethnic-specific recommendation for carrier
screening. The number of screening tests will be chosen based on the couples’
ethnicity as people in a different ethnicity will have genetic susceptibility or
mutation for a particular disease. For example, Tay Sachs disease (TSD), an
autosomal recessive degenerative neurological disease, will be screened in the Jewish
disease screening, as it is one of the common inherited diseases among the Ashkenazi
Jewish (AJ) population. Basically, this approach needs a reliable family and ascertainment
of ethnicity2, 3. It may be more difficult to identify the patients’
ethnicity as they tend to have a mixed ethnic background with or without their

Leading to new patterns of carrier screening,
pan-ethnic screening and expanded carrier screening, that extends screening for
all diseases in all patients regardless their ethnicity. The different between
pan-ethnic screening and expanded carrier one is the number of the test. With
expanded carrier testing, there is only one screening panel. Although this test
is presumably available and easy to perform by testing the couples’ blood
sample, the detection rate, sensitivity and residual risk for the general
population are difficult to estimate2, 3 Thus, the utilisation of this test should
be under genetic counselors, as it can identify a wide range of genetic
diseases, both known to be severe diseases affecting children’s quality of life
and the rare ones with various phenotypes. In addition, there may be some
rarer mutations which cannot be detected with current carrier screening tests. If an appropriate genetic counseling cannot be provided, the results
of this test would increase the couples’ anxiety with misinterpretation, as
well as waste their money and time on further tests29.

Another fascinating innovation involving prenatal
screening and diagnosis is preimplantation genetic diagnosis (PGD) which is
primarily used in infertility clinics to evaluate preimplantation embryonic
cell for chromosomal abnormalities and other molecular anomalies. Its objectives
are to reduce the risks of having a child with inherited genetic diseases,
decrease failure rate of implantation, and pregnancy losses in the couples with
problems of male infertility, advanced maternal age, and
recurrent miscarriage. In the genetic screening point of view, it also reduces
the rate of elective abortion in a potentially affected child30. If
this innovation was applied to routine practice in the general population,
every couple would Ideally have healthy children without any genetic diseases.

However, it would not only interrupt the normal conceptual process but also consume
significant resources.

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