J. Perinat. Med.

2014; 42(5): 545–548

Academy’s Paper

Giovanni Monni*, Maria Angelica Zoppi, Ambra Iuculano, Alessandra Piras and Maurizio
Arras

Invasive or non-invasive prenatal genetic

diagnosis?
DOI 10.1515/jpm-2014-0135
fetal malformations or other abnormal ultrasound find-
ings. The initial policies led to an offering of invasive
prenatal diagnosis to 5% of all pregnant women (positive
About 40  years ago, invasive prenatal diagnosis tech-
screen rate), with a 40% detection rate for trisomy 21.
niques were introduced in obstetrics. Initially, amnio-
With the advent of biochemical screening tests using
centesis was performed followed by placentacentesis,
maternal serum in the second trimester, the “triple”
fetoscopy, fetal blood sampling (FBS), and chorionic
and “quadruple screen”, the detection rate of trisomy 21
villus sampling (CVS). These procedures, while invad-
increased to 60% [28]. Meanwhile, with advancements
ing the uterine environment, have made it possible to
in ultrasound, numerous reports were published that
proceed with the retrieval of biological tissue of fetal
identified sonographic “markers” for trisomy 21, leading
origin for analysis and definitive diagnosis [6, 20, 21, 27].
to additional screening with a “genetic ultrasound”
The development of such techniques was facilitated by
in the second trimester [1]. However, second trimester
improvements in instrumentation and technology, and
ultrasound for the purposes of screening an unselected
was further propelled by the advancement of cytogenetics
population never gained universal acceptance, and was
and molecular genetic techniques [3, 13, 14, 22].
primarily used in higher-risk populations (i.e., a woman
However, invasive prenatal diagnosis carries the
with advanced maternal age that wished to avoid inva-
inherent risk of fetal loss, which is low, but not negligi-
sive testing). At this time, second trimester amniocentesis
ble (amniocentesis and CVS approximately 0.3%–0.5%
was the primary invasive diagnostic test practiced, while
and FBS 1%–2%). In addition, there is a significant eco-
fetal blood sampling by cordocentesis was utilized when
nomic burden from the costs associated with laboratory
a diagnostic test was desired later in the second trimester,
techniques. Public health programs of nations interested
often in the setting of identification of a fetal anomaly in
in the utilization of invasive procedures have generally
the second trimester ultrasound. As mean maternal age
limited their use to high-risk cases, where the risk of pro-
at childbirth continued to increase, especially in Western
cedure related loss is comparable to the risk of an affected
countries, alongside increasing scientific advancements,
fetus for a given condition [25, 26]. As a result, in the
the number of indications for prenatal diagnosis rose.
1980s amniocentesis was offered to women at higher risk
This trend led to an increased rate of invasive prenatal
of trisomy 21 based on maternal age alone, if there was an
diagnosis, based on maternal age alone up to 15–20% in
increased risk due to prior complications or pre-existing
some nations, and spurred a search for new solutions.
conditions (i.e., chromosomal alterations in the parents
In the mid-1990s, an important turning point was the
or in prior offspring), or because of prenatal detection of
use of ultrasound to measure fetal nuchal translucency
at 11–14 weeks [17], along with maternal serum biochemi-
cal screening, pregnancy associated plasma protein-A
*Corresponding author: Giovanni Monni, Department of (PAPP-A), and free beta subunits of human chorionic gon-
Obstetrics and Gynecology, Microcitemico Hospital, Prenatal and adotropin (free beta-hCG), for the screening of trisomy 21
Preimplantation Genetic Diagnosis, Cagliari, via Edward Jenner SNC, [24]. With a similar invasive testing rate as maternal age
Sardinia, Italy, Tel.: +39-706095546, Fax: +39-706095514, alone of 5%, the combined test led to a detection rate of
E-mail: prenatalmonni@tiscali.it
approximately 90% for trisomy 21 (in the setting of a posi-
Maria Angelica Zoppi, Ambra Iuculano, Alessandra Piras and
Maurizio Arras: Department of Obstetrics and Gynecology,
tive test, the odds of an affected fetus were 1:24). The intro-
Microcitemico Hospital, Prenatal and Preimplantation Genetic duction of the nuchal translucency measurement raised
Diagnosis, Cagliari, via Edward Jenner SNC, Sardinia, Italy the issue of certification, reproducibility, and reliability of
546      Monni et al., Invasive or non-invasive prenatal genetic diagnosis?
measurements. Additionally, it helped launch the positive
and ambitious process of ultrasound accreditation, which
served as a reference for many other systems of accredita-
tion in obstetric ultrasound (however, it was criticized for
the level of cost and difficulty) [15].
First trimester screening and the measurement of
nuchal translucency, in particular, changed the course of
prenatal care. From the latter half of the 1990s, obstetric
guidelines of many nations placed the 11–14 weeks ultra-
sound as a routine part of prenatal care. The importance
of this evaluation was further reinforced after multiple
reports demonstrated an association between an abnormal
nuchal translucency and major congenital malformations,
especially cardiac defects [23]. Additional benefits from the
first trimester ultrasound that were explored included early
recognition of major fetal malformations, or their early
exclusion. After the passage of time, however, it must be
admitted that ultrasound in the first trimester never gained
widespread recognition for anatomical assessment [8].
Many critical issues in first trimester ultrasound remain
unresolved, most importantly, that fetal structures are
often too small or undeveloped for proper evaluation (little
of which is ameliorated by the transvaginal approach).
Furthermore, assigning a pathologic diagnosis to a global
abnormality is complicated because the etiology may not
be discovered until later in gestation (e.g., disproportion of
the cardiac chambers). A definitive diagnosis is difficult to
come by and pathological anatomy analyses cannot be per-
formed until later in pregnancy. Indeed, the utility of per-
forming early ultrasound anatomical screening has been
questioned because such screening rarely offers a com-
prehensive diagnosis, and abnormal findings only serve to
raise anxiety of both practitioners and expecting parents.
In cases of abnormal nuchal translucency measure-
ment, complex algorithms have been devised for prenatal
care. Included in most algorithms are assessment of fetal
karyotype, early echocardiography and early second tri-
mester sonographic assessment of fetal anatomy, repeat
sonographic assessment later in the second trimester, and
neonatal follow-up. The increasing use of nuchal translu-
cency screening, and of the combined test, has reduced
the importance of genetic ultrasound in the second tri-
mester and replaced the use of the triple or quadruple
screen unless risk screening was not performed in the first
trimester. Concurrently, elaborate programs for second tri-
mester testing were suggested, such as the integrated test,
which involves the questionable policy of non-disclosure
until the second trimester [5, 29]. However, integrated
testing did provide the unequivocal benefit of reducing
the screen-positive rate to 1%, while maintaining a sensi-
tivity around 90%.
In regards to invasive prenatal diagnosis, the identifi-
cation of cases at greater risk of aneuploidy by first trimes-
ter screening led to a general rate of decline in invasive
procedures, thereby reducing both the economic burden
and the number of fetal losses. Another effect of first tri-
mester screening was the shift in prenatal diagnosis from
the second to the first trimester. As a result of the greater
predictive value of first trimester screening, compared to
the maternal age alone, the request of prenatal diagnosis
by CVS increased [18, 31]. Hence, an important conse-
quence was the greater demand for CVS and those trained
to perform the procedure. The number of practitioners,
once performing predominantly amniocentesis, who have
had to improve in CVS, has risen consistently over the last
number of years. As a result, educational organizations
have faced new challenges in providing such training [11].
The effects of introducing the combined test went
beyond the single issue of screening for trisomy 21 and
created the recently expressed concept of reversing the
traditional prenatal care pyramid [16]. Some additional
information can be obtained by collecting other feto-pla-
cental biochemical markers (i.e., PlGF Placental Growth
Factor), placental growth factor). However, it remains to
be shown that this can translate into more effective moni-
toring and intervention for certain complications of preg-
nancy, such as spontaneous abortion, preterm delivery,
preeclampsia, intrauterine growth restriction, and gesta-
tional diabetes.
In addition to the developments discussed above
in the 1990s, pre-implantation genetic diagnosis (PGD)
technology became available and allowed couples that
are carriers for, or affected by, genetic diseases to select
unaffected embryos for transfer before implantation, after
in vitro fertilization (IVF) [10]. The option of PGD offered
the opportunity to circumvent wrestling with the option of
pregnancy termination in affected fetuses [12]. The prac-
tice of PGD has evolved rapidly, in terms of both diagnos-
tic techniques and biopsy methods. Indeed, testing for a
specific gene mutation can be performed in concert with
24-chromosome aneuploidy screening [7]. However, this
technique has also stirred much ethical debate about the
interests of the prospective parents, the embryo, the child,
and the people affected by these diseases.
In the field of prenatal invasive diagnostic tech-
niques, there was no major news for many years. Recently,
however, two developments have appeared, and they
will likely cause a great stir. These techniques are the
array comparative genomic hybridization (aCGH) or chro-
mosomal microarray (CMA) in the field of the invasive
prenatal diagnosis, and in the field of the screening, non-
invasive prenatal tests (NIPT) [4, 30].
 Monni et al., Invasive or non-invasive prenatal genetic diagnosis?      547
Comparative genomic hybridization has emerged in
recent years as a primary diagnostic tool for the evalu-
ation of developmental anomalies and structural mal-
formations in children. Chromosomal copy number
variation (CNV), detectable by aCGH, is a variation from
the expected number of DNA segment copies when com-
pared to a reference genome.
aCGH allows detection of smaller pathogenic chro-
mosomal variants that are undetectable using standard
cytogenetic analyses. An important aspect is the signifi-
cant frequency of aCGH findings in fetuses with particu-
lar structural abnormalities, and a lower but still notable
frequency of aCGH abnormalities in cases with other find-
ings, such as fetal hydrops, cystic hygroma, and abnormal
nuchal translucency. Although currently quite expensive,
this technology has the advantage of being automated,
requiring less personnel and providing faster turnaround
time. Of note, the traditional G-banding technique used in
aCGH does not detect balanced chromosomal rearrange-
ments, triploidy, and some instances of mosaicism. The
biggest clinical challenge presented by aCGH is the detec-
tion of chromosomal variants of unknown clinical signifi-
cance (VOUS) [19]. Currently, there are two policies about
the implementation of the aCGH analysis in prenatal
diagnosis: the first, more traditional approach, suggests
aCGH only in cases with specific indications such as fetal
malformations, and the second approach proposes aCGH
as an alternative, and not necessarily as a replacement,
to traditional karyotype screening even in low-risk cases
[30]. There is a general consensus on the need for proper
genetic counselling prior to performing this test.
The second recent advancement is the use of cell free
fetal DNA (cff-DNA) in maternal blood for non-invasive
prenatal testing [4]. Non-invasive prenatal diagnosis for
fetal sex determination, RhD antigen, paternal inherited
genes or some de novo autosomal dominant diseases
have been validated but there are many concerns about
non-invasive prenatal diagnosis for monogenic diseases
(autosomal recessive diseases, X linked diseases, and
autosomal dominant diseases of maternal origin) [2].
Regarding autosomal trisomies, such as trisomy 21,
18, 13, sex chromosome aneuploidies, and triploidy, NIPT
is employed mostly using shotgun massively parallel
sequencing (s-MPS) [2]. For trisomy 21, individual studies
showed the detection rate (DR) ranges between 94.4% and
100%, and false positive rates (FPR) ranges between 0 and
2.1% [9]. Fairly good results have been demonstrated for
detection of trisomy 18, while screening for trisomy 13 and
monosomy X has a poorer performance due to the highly
variable amplification of these chromosomes with lower
guanosine and cytosine content. The main limitations of
NIPT for chromosomal abnormalities are the frequency
of failure to provide results (1–5% of cases), the delay in
obtaining the results (1–2 weeks), and the high cost of the
technique.
The expectations regarding cff-DNA for fetal genetic
anomalies are very high, as it may have the potential to
change the landscape of prenatal diagnosis. However, to
the disappointment of many, cff-DNA does not have the
ability to function as a diagnostic test but is considered a
“super” screening test. Use of NIPT has been proposed for
use as either screening for the general obstetric popula-
tion or as a contingent screening test in high-risk groups.
The first option would be quite expensive given the sheer
volume of tests performed and the subsequent invasive
testing required for positive cases (number of false posi-
tives expected is about 1%) [9]. However, this option would
maximize the prenatal detection rate (99%). The second
approach implies a lower number of invasive procedures
(0.4–0.8%) with a very high detection rate (86–97%),
depending on how the “high-risk” group is classified. Kar-
yotype in the high-risk group can alternatively be obtained
by the QF-PCR method, which has the advantage of offer-
ing very rapid results (useful in cases where the likelihood
of abnormal karyotype is very high). It can also be studied
by aCGH methods, whose rationale is intuitive, in cases of
malformations or with an enlarged nuchal translucency
and normal karyotype. At present, a few groups are study-
ing implementation of NIPT after the use of first trimes-
ter screening. Under these circumstances, a position of
watchful waiting appears reasonable.
The scientific community must carefully consider the
economic and ethical issues of NIPT and the impact that it
may have on well-established methods of prenatal screen-
ing and diagnosis [2]. However, for those who have the
duty of this consideration, should reflect on the fact that
modern society is ever more web-based and that the web,
rather than the counselling that geneticists can offer, may
be the primary, although misleading, source of informa-
tion for many prospective parents.
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The authors stated that there are no conflicts of interest regarding
the publication of this article.