Prenatal diagnosis: choose between invasive, non-invasive and novelty analyses

Prenatal diagnosis includes a series of techniques, both invasive and non-invasive, which make it possible to diagnose the fetus at different times of pregnancy and understand if there are genetic abnormalities in the baby.

Let’s find out together what invasive technique means, which diseases can be discovered, which risks they entail.

It differs from pre-implantation diagnosis, which is also considered the earliest prenatal diagnosis technique, since this is performed on the embryo before its transfer into the uterus.

Invasive and non-invasive

Prenatal diagnostic techniques can be invasive, i.e. made by taking (usually with a needle) tissue or amniotic fluid, or non-invasive because they are limited to blood analysis without involving risks for the fetus.

Based on their invasiveness we can distinguish:

1. Non-invasive analysis : circulating fetal DNA and combined test
2. Invasive analyses : celocentesis, CVS , amniocentesis , cordocentesis

The material taken is then subjected to laboratory analyses, which can last even a few days. There are different techniques that can be applied, more or less complex, and which allow to diagnose different anomalies.

Based on the laboratory techniques used for diagnosis, we can distinguish:

1. Standard karyotype
2. QF-PCR
3. CGH-ARRAY
4. Analysis of monogenic diseases

Prenatal diagnosis: non-invasive tests

Non-invasive prenatal analyzes are risk-free even if they allow a more limited diagnosis both in precision and in the number of diagnosable anomalies.

Circulating fetal DNA test

The circulating fetal DNA test is an innovative non-invasive diagnostic technique that allows you to analyze the small amount of fetal DNA present in the mother’s bloodstream.

The analysis is performed by means of a simple blood sample from the mother performed at the 9th-10th week of pregnancy.

This technique, recently introduced in Italy as well, is currently still very expensive and therefore difficult to propose on a large scale.

The diagnostic technique with a sensitivity of 99% the most common fetal chromosomal anomalies linked above all to advanced maternal age, i.e. trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), anomalies of sex chromosomes (Turner syndrome, Klinefelter syndrome, etc.).

ADVANTAGES : being a non-invasive technique, it eliminates the risk of fetal loss.

DISADVANTAGES : it has a high cost since it was recently introduced and it analyzes only 5 chromosomes even if with maximum sensitivity.

Combined test or ultrascreen or ultratest

This non-invasive test, also called ultrascreen or ultratest , provides a statistical risk calculated thanks to the dosage of two hormones (fbHCG and PAPP-A) present in the maternal blood in the third month of pregnancy and the ultrasound measurement of some fetal parameters, the most important of which is the nuchal translucency (measurement of the physiological collection of liquid present in the region of the nape of the fetus).

The test has a sensitivity of 90-96%, therefore with a high percentage of false positives (high risk test result but healthy fetus) and false negatives (low risk test result but affected fetus).

False positives cause an increase in “useless” invasive prenatal diagnoses (chorionic sampling and amniocentesis), while false negatives can cause the birth of a fetus with chromosomal abnormalities.

The result of the test is a risk value of fetal pathology, considered “low” if less than 1/350 or “high” if greater than 1/350.

In the event of a “high-risk” result, after a further second-level ultrasound check, the pregnant woman can be directed towards an invasive prenatal diagnosis.

In any case it is a calculation of the presumed risk and in no case with this test can we know the chromosomal constitution of the fetus.

Furthermore, the test does not give any information about the countless genetic diseases caused by an anomaly of a single gene (about 6,000,000).

According to the most recent guidelines of the Fetal Medicine Foundation (FMF), it is advisable to separate the sampling from the ultrasound, and therefore perform the hormone dosage between the 9th and 10th week of pregnancy, while performing the ultrasound between the 11th and the 12th. This allows to increase the sensitivity of the test up to 95-96%, if the instrument used for the hormone dosage, the operator who performs the ultrasound and the software which performs the analysis are FMF certified, otherwise the test has poor reliability.

ADVANTAGES : being a non-invasive technique, it eliminates the risk of fetal loss. It has a very low cost.

DISADVANTAGES : poor sensitivity, especially if performed in a non-compliant manner (non-FMF accredited centres). Often, pregnant women with normal fetuses are referred for invasive prenatal diagnosis.

Prenatal diagnosis: invasive tests

Coelocentesis

Coelocentesis is an invasive method that is performed around the 8th-9th week of pregnancy in highly specialized centers (only 2 in Italy), by which, via the vaginal-cervical route, under ultrasound guidance, a small quantity of coelomic fluid from the embryonic coelomic cavity.

From this liquid the DNA is then extracted for the genetic analyzes of the case.

The technique has a double risk of fetal loss compared to CVS , quantified at about 1.5-2%.

At present, the diagnosis of aneuploidies of 5 chromosomes (13, 18, 21, X, y) and the diagnosis of a person suffering from thalassemia (Mediterranean anemia) are performed on coelomic DNA, but the technique is virtually applicable to all genetic diseases.

The high risk of fetal loss justifies its use only in the presence of couples with a known genetic disease transmissible to the offspring .

Finally, there is a small risk of maternal-fetal contamination which can invalidate the result of the analysis, but in general the reliability is very high (98-99%).

ADVANTAGES : earliness of the diagnosis which, in case of positivity, allows a very early termination of pregnancy.

DISADVANTAGES : technique performed only in very few centers in the world; higher risk of fetal loss.

CVS

CVS is an invasive method that is performed around the 10th-13th week of pregnancy, by means of which, transabdominally, under ultrasound guidance, a small quantity of chorionic villi is taken, i.e. the embryonic part of the placenta, from which then the karyotype analysis is carried out and, if required, also the molecular analysis for specific genetic disease.

The technique has an estimated risk of fetal loss of around 1%.

For standard karyotype analysis, the villi are both analyzed directly (direct analysis, with 5 days report) and cultured (long-term culture, with 20 days report). Many centers that offer CVS combine it with QF-PCR (see below) with a report in 2 days. It is always advisable to combine the chorionic villus analysis with the exclusion of maternal contamination, which is performed by taking a small amount of buccal mucosa from the mother at the same time as the chorionic villus sampling.

Amniocentesis

Amniocentesis is an invasive method which is performed around the 16th week of gestation by which a small quantity of amniotic fluid is taken transabdominally, under ultrasound guidance, on which the karyotype analysis is carried out and, if required, also molecular analysis for specific genetic disease. The technique has an estimated risk of fetal loss of around 0.5%. For the standard karyotype analysis, the amniocytes (amniotic fluid cells) are cultured and analyzed after at least 2 weeks (report in 20 days). Many centers that offer amniocentesis combine it with QF-PCR (see below) with a report in 2 days.

Cordocentesis

It is an invasive method that is performed, only for specific indications, around the 18th-20th week of gestation by which, by trans-abdominal route, under ultrasound guidance, a small amount of fetal blood is taken from the vessels of the cord umbilical , on which the specific confirmatory analyzes that required the use of this technique are carried out.

The technique has an estimated risk of fetal loss of around 1% if the procedure is performed after 20-22 weeks, while it is more risky if it is performed at 16-18 weeks.

Not all centers perform this procedure as it requires great experience and dexterity to minimize the risks.

The same analyzes of postnatal age can be performed on fetal blood, including infectious disease tests.

Advice in case of invasive prenatal diagnosis

In the event of an invasive procedure, it has now become routine to prescribe a short 3-day antibiotic therapy to be taken in conjunction with the sampling, in order to drastically reduce the risk of iatrogenic fetal infections (caused by the procedure).

The prescribed antibiotic does not harm fetal development, so it is a good idea to make sure that the gynecologist performing the procedure prescribes it.

Finally, it is appropriate to ensure that the invasive diagnostic procedure is performed in the operating room or in any case in a sterile environment , with the exclusive use of sterile disposable material, in order to minimize the risk of infection.

It is good practice that, after an invasive procedure, the pregnant woman remains at rest semi-seated for at least an hour and that a control ultrasound is performed before being discharged to verify the good health of the fetus and placenta.

Laboratory techniques in prenatal diagnosis

The main techniques used for prenatal diagnosis are:

1. Standard karyotype
2. QF-PCR
3. CGH-ARRAY
4. Analysis of monogenic diseases

Standard karyotype

The analysis of the karyotype (or also called chromosome map) allows the 46 chromosomes to be visualized from a numerical, morphological and structural point of view. The analysis is performed both on chorionic villi and on amniocytes, and can be either direct, provisional (through the analysis of analyzable cells present in the fresh sample; reporting time 3-5 days), or after long-term culture (possibility of counting many cells, with certainty of the final result; average reporting time 21 days).

This analysis allows to visualize gross alterations of the chromosomes (in number and structure) but not the small alterations (small deletions or duplications) nor the anomalies of the single genes.

QF-PCR

The analysis performed using the fluorescent quantitative PCR technique (QF-PCR), i.e. the analysis of the 5 chromosomes (13, 18, 21, X, Y) most involved in chromosomal abnormalities from advanced maternal age, can be performed both on chorionic and on amniocentesis, and is generally proposed in combination with the standard technique, to quickly provide (in 2 days) a partial result followed by the definitive one after 21 days. The technique gives only rough information on the number of the 5 chromosomes analyzed and no information regarding the structural anomalies, the anomalies of the number of the other 18 chromosomes, the small deletions or duplications nor the anomalies of the single genes.

CGH-ARRAY

CGH-array analysis allows performing a complete molecular karyotype in 3 days, both on villi and amniocytes, with a definition about 100 times higher than a standard karyotype. The report is conclusive. There are three types of CGH-arrays (BAC, Oligo and SNP) which have increasing definition and precision (from BAC, less detailed, to very detailed SNP). It allows the identification of chromosomal alterations in number and structure (unbalanced, and therefore pathological) and small deletions and duplications (about a hundred of which are involved in genetic malformation syndromes or with psychomotor retardation).

It does not identify balanced translocations (non-pathological in 99.5% of cases) and abnormalities of single genes. The technique is highly recommended in cases of suspected ultrasound anomaly.

Analysis of monogenic diseases

Both on chorionic villi and on amniocytes it is possible to perform, generally through direct sequencing (if the causative mutation is known) or analysis of the familial haplotype (if the involved chromosomal region is known but not the disease gene), the analysis of single monogenic diseases. The correct procedure provides that these analyzes are performed on specific indications (family disease, parental carrier status, precise ultrasound suspicion) after having characterized the parents from a molecular point of view in the pre-natal period.

The “screening” analysis (without specific indication) of a few genetic diseases in the prenatal period does not make any sense, since there are about 6500 genetic diseases and the exclusion of a dozen of them does not change the risk of fetal genetic disease .