Invasive Diagnostic Procedures

Invasive Diagnostic Procedures

Florencia Petracchi

Silvina Sisterna


Prenatal diagnostic procedures allow the detection of an increasingly broad list of fetal anomalies. By obtaining a sample of chorionic villi, amniotic fluid, or fetal blood, genetic and biochemical tests can be performed. The objective is to determine whether the fetus has a specific disease. As a result, patients and their physicians can be provided with useful information to allow for pregnancy management.

The benefits of performing prenatal tests include reassuring patients when the results are normal, identifying disorders for which prenatal treatment can provide benefits, optimizing neonatal outcomes (ie, selecting the more adequate hospital and neonatology service for the reception of the affected child), and considering termination of pregnancy.

The prenatal diagnosis gold standard is obtained through the analysis of different fetal or placental tissues: chorionic villus (CVS for chorionic villus sampling), amniotic fluid (for an amniocentesis, AC), or blood (for a cordocentesis, CC).

Over the last few years, a significant advance in prenatal diagnosis has occurred, especially regarding genetic tests. In the early years of prenatal diagnosis, prenatal genetic tests focused on testing for Down syndrome (trisomy 21). Currently, a greater number of diseases can be detected. However, prenatal genetic tests cannot identify all genetic abnormalities present in a fetus. The scope of different tests; the detection rate; false-positive rate; incidental, conflicting, and unknown findings; the techniques; and the risks and benefits should be addressed before any patient undergoes an invasive prenatal diagnosis procedure. This task can be achieved through genetic counseling (see Chapter 11).

On the one hand, pretest counselling should include information about the risks, the scope, and the limitations of the studies; concerns and fears of the patients and the different options after the results should be addressed. It is important that patients understand the benefits and limitations of screening and prenatal diagnosis tests, including the diseases that may or may not be detected.

On the other hand, the wide range of clinical presentations or phenotypes for many genetic disorders should be explained as well as the fact that the results of molecular tests would not always be able to predict the exact phenotype in the child.

Although the scope of prenatal genetic diagnosis is usually based on the identification of fetal karyotype anomalies (through conventional cytogenetics or chromosomal microarray), it is also possible to perform other analyzes such as specific genetic mutations and gene panels or complete exome sequencing (Table 14.1).

The objective of this chapter is to describe the main aspects of invasive fetal procedures for prenatal genetic diagnosis. Genetic counseling, technical aspects, clinical indications, diagnostic capabilities, and possible complications are explained.

Pretest Genetic Counseling

Before an invasive prenatal procedure is carried out, genetic counseling should be provided by trained professionals. Genetic counseling begins with the recollection of both personal and family history including ethnicity, obstetric/gynecologic history, medical and surgical history, genetic and acquired disorders/diseases, and lifestyle and habits. These variables that can increase the fetal risks are summarized in Table 14.2. The aneuploidy age-related risks at different gestational ages should be discussed. It is recommended to talk about personal values and the expectations of the pregnant woman and her family. In some cases, it may be necessary to ask for a parental karyotype due to, for example, family or personal history of recurrent pregnancy
losses, fetal deaths, or fetal malformations. Less frequently, it may be necessary to carry out a clinical genetic evaluation when one of the parents is suspected to have an undiagnosed genetic syndrome or any other member of the family is suspected to or has been diagnosed with a genetic condition.

When couples are informed of the risks of having a child with birth defects or a genetic disorder before pregnancy, they will have the possibility to choose between different reproductive options: contraception, gamete donation, adoption, preimplantation genetic testing (PGT), prenatal screening, or diagnostic testing or newborn testing.

Genetic tests should be discussed before pregnancy or in the first obstetric visit. The attending physician should provide a clear explanation in an appropriate level for their education, literacy, and language skills. The risk of aneuploidies and other genetic diseases and the differences between screening and diagnostic tests should be explained. Background risks of fetal loss should also be addressed (Algorithm 14.1).

Once the decision of performing an invasive procedure is taken, counseling should include a verbal description and, if possible, illustrate with diagrams or images the most appropriate prenatal procedure. The topics that should be discussed during pretest counseling are summarized in Table 14.3. Pretest counseling should be a shared decision-making process, where the health provider exposes complete information and offers all available options so that the couple can take the
best-informed individual choice. Informed consent should be provided and signed back before the invasive test is carried out.

Prenatal Diagnostic Tests

There are different techniques to obtain samples for prenatal diagnostic tests: CVS, amniocentesis, and cordocentesis (fetal blood sampling) (Table 14.4; Figures 14.1, 14.2, 14.3, 14.4). Fetal tissue sampling may be necessary in very few cases.

Chorionic Villus Sampling

First described in China in the mid-1970s, CVS was introduced into clinical practice in the early 1980s1,2 and consists of obtaining chorionic villi for cytogenetic or molecular test of trophoblastic cells from the placenta.


Chorionic villi can be obtained by transabdominal or transcervical approaches. The procedure is performed under continuous ultrasound guidance.

First, an ultrasound scan is performed to determine gestational age, fetal heart rate and anatomy scan, the position of the chorion and the best approach is decided.

The tip of the needle or catheter used is placed in the placenta without entering the amniotic cavity.3 The aspiration of the chorionic villi is performed through the negative pressure generated by the syringe, either through a “hands-free technique” or using a biopsy adapter. Because there is a lack of data comparing the safety and efficiency of these two methods, the choice must be made according to the experience or preference of the physician.4,5

Placental villi may be obtained through transcervical or transabdominal access to the placenta. Fetal loss and successful sampling are similar for both routes.6

  • Transabdominal CVS: Local anesthesia can be applied. A 17- to 20-gauge (G) needle or a needle with 17/19-G outside and 19/20-G inside can be used.7 Once the needle has reached the target within the placenta, between one and 10 back-and-forth movements are performed, while the vacuum is maintained and the sample is aspirated manually by an assistant or with a vacuum adapter.5,8

  • Transcervical CVS: A plastic catheter with a metal stylet connected to a syringe is used to make the aspiration. First, a speculum is inserted into the vagina, and then the vagina and cervix are cleaned with an antiseptic solution. Using ultrasound guidance, the catheter is placed in the cervical canal until it reaches the chorion, from where the sample is suctioned through the tube
    into the syringe.9The number of villi obtained should be verified visually. A minimum amount of 5 mg of villi is required in each sample to achieve a valid result.9 After the procedure, an ultrasound scanning is recommended to check for fetal heart rate.

Gestational Age

CVS should not be performed before 10 full weeks of gestation, due to the increased risk of fetal loss and complications.4,10 There is an increased risk of limb reduction anomalies and oromandibular hypoplasia in fetuses when CVS is performed earlier than
10 weeks of gestation. However, there is insufficient evidence to confirm the causality of this observation, and posterior studies have shown no differences between exposed and nonexposed fetuses.11,12

Laboratory Aspects


Chromosomal microarray analysis (CMA) can identify major chromosomal aneuploidies as well as submicroscopic changes that cannot be detected by conventional karyotyping and can be performed either directly on uncultured tissue or on cultured cells.

Most prenatal diagnosis clinics in the world have switched to using microarray as the first test whenever a prenatal invasive diagnosis is required (Algorithm 14.2). Failure of obtaining a result in a prenatal microarray is around 1%.13 CMA can detect a pathogenic (or likely pathogenic) copy number variant in approximately 1.7% of patients with a normal ultrasound examination result and a normal karyotype. Therefore, CMA is recommended to be made available to any patient choosing to undergo invasive diagnostic testing.3


The errors reported in CVS results may be due to maternal cell contamination, culture artifacts, or placental and fetal mosaicism.

Maternal Cell Contamination

Maternal cell contamination can occur when maternal cells are cultured instead of cytotrophoblastic cells. In CVS, this happens in 1.7% to 4% of cases. Therefore, separating decidua and chorionic villi (CV) with a dissecting microscope before processing the sample is recommended. Different studies can be performed to detect maternal contamination, especially if molecular diagnostic tests are performed.14


Mosaicism is the presence of two or more cell lines in the same tissue. It represents different chromosomal complements in the fetoplacental unit developed from a zygote. Chromosomal mosaicism is a consequence of a viable somatic postcigotic error.

The placental mosaic pattern depends on several factors such as the moment of the mutational event, the affected cell lineage, cell viability, and selection.

Types of mosaicisms include (Table 14.5)16

  • Confined placental mosaicism (CPM): a chromosomally abnormal cell line exists in the extraembryonic tissues of a normal embryo 46,N (euploid). This type of mosaicism is only diagnosed by CVS and not by amniocentesis.18

  • True constitutional mosaicism: mosaicism is present in all fetal cells.

  • Pseudomosaicism: an anomaly that arises during tissue culture (cultural artifact), but the embryonic and extraembryonic tissues are all 46,N.

If an abnormal cell line is detected with a normal 46,XX (or 46,XY) cell line, different types of mosaicism can be recognized, depending on the combination of the tissues containing the affected cells.

Mosaicism of placental cells is observed in 1% to 2% of the CVS. As the exact embryo-fetal developmental stage in which the mitotic error occurs cannot be established with certainty with karyotyping, the retrieval of a mosaic in CVS does not necessarily imply a fetal involvement (true fetal mosaicism, TFM) as it could be restricted to the placenta (CPM). For this reason, in this situation, a confirmatory karyotype using amniocentesis is recommended to discriminate a generalized mosaicism (placenta and fetus affected by the abnormal cell line) from a confined mosaicism (only in the placenta affected but not the fetus).

Genetic counseling is strongly recommended, and amniocentesis and/or cordocentesis may be necessary. Besides the clinical reassessment at ultrasound, before a confirmatory amniocentesis is performed, women should be counseled about the likelihood that the specific abnormal cell line in CV could also be extended to the fetus (TFM). The results can be conflicting for the parents, because it is important to differentiate TFM from confined placenta mosaicism, which both carry different prognoses. It is uncommon that an observation of apparent CPM at CVS reflects a true constitutional mosaicism of the fetus.

TFM is observed infrequently, and most chromosomal mosaicisms identified at prenatal diagnosis, more especially in CVS, do not presage an abnormal fetus.16

Management of Pregnancy Amniotic fluid sampling is considered the standard procedure of confirmation because it provides cells for cytogenetic analysis coming mainly from fetal anatomical structures, including the urogenital tract, the respiratory apparatus, and the epithelial system representing different embryological layers.19,20

If the mosaicism is found only in short-term culture, it usually has a mitotic origin and it is more likely to be CPM or pseudomosaicism. However, when mosaicism is present in both short- and long-term cultures, it is more likely to be found in the fetus.20

Based upon an analysis of 67,030 CVS samples, Grati et al20 reported that mosaicism occurred in 2.17% of the cases, and of these, just 13.5% (0.22% of the total CVS samples) proved to have a TFM.

Mosaicism in Array Studies CV tissue culture is no longer conducted in many laboratories, primarily because of the labor-intensive procedure of requiring both short and long term cultures. Instead, microarrays are frequently performed on intact CV tissue that includes analysis of the cytotrophoblast layer. CMA can detect mosaicism at levels as low as 9%. Among prenatal CMA of CV, the incidence of mosaicism seems to be similar to conventional chromosomal tests (3%). A study conducted by Gu et al21 showed that mosaicism can involve not just entire chromosomal abnormalities, but submicroscopic copy number variation (CNV) and mosaic aneuploidy plus mosaic CNV. As the analyzed tissue is cytotrophoblast layer, when a mosaicism is found by CMA, a confirmatory test should be completed using CV long-term culture and amniotic fluid. Approximately half of mosaicisms found by CMA were CPM based on a normal confirmatory amniotic fluid result and/or normal findings on long term cultured CV cells. This rate is lower than previously reported rates based on karyotype analysis, but this may be due to the small sample size of the study.21

Imprinting and Uniparental Disomy

Imprinting means that the activity or nonactivity of a gene (or chromosomal segment) depends on the parental origin of the chromosome upon which the gene (or segment) is located. Thus, a chromosomal segment can receive an “epigenetic mark” or is “imprinted,” as is transmitted from parent to child, depending upon whether it is the mother or the father who has contributed that chromosomal segment.16

Uniparental disomy (UPD) occurs if both segments originate from one parent, resulting in either double the amount of expression (biallelic) or no expression (nulliallelic), according to the gender of the contributing parent. If this is a functional imbalance, it will cause a phenotypic effect in the offspring.16

UPD has been observed for every chromosome except 19.22 Major syndromes related to UPD are Prader-Willi syndrome and Angelman syndrome (chromosome 15), Beckwith-Wiedemann syndrome (chromosome 11), Silver-Russell syndrome (chromosome 7), transient neonatal diabetes (chromosome 6), maternal UPD 14 (Temple syndrome), and paternal UPD 14. However, for most chromosomes, there is no apparent phenotypic consequence.

Complications of CVS

Fetal Loss

Evidence on the risk of miscarriage related to the procedure comes from historical retrospective cohort studies. The risk of fetal loss compared to controls varies between 0.2% and 2%.4,23 This risk seems to be lower in centers with broad experience in prenatal diagnosis, ranging between 1/150 and 1/500.4,24

A retrospective study of the Danish registry, examining 31,355 cases of CVS, reported a total fetal loss rate of 1.9% (vs 1.4% with amniocentesis); the rate of spontaneous abortion was inversely proportional to the number of procedures performed and was 40% higher for centers that performed fewer than 1500 procedures per year compared with those that performed more than 1500 per year.25 An update in 2016 from the same database reported that there was virtually no impact of CVS on fetal loss rates (risk of spontaneous abortion, 0.21% at 21 days after CVS).26 This result is similar to the findings of a retrospective study that calculated the procedure-related risk as a risk-difference and concluded that the estimate risk of procedure-related loss was 0.29% for CVS.27

According to a recent review of the literature and meta-analysis, the rate of fetal loss due to CVS does not seem to increase significantly compared to the unexposed population. The reported risk was of 0.20% (95% CI, −0.12 to 0.52; I2 = 51.9%) (Table 14.6).28

A large randomized controlled trial (RCT) conducted by Smidt-Jensen compared transcervical and transabdominal CVS and concluded that the risk of pregnancy loss is similar for both procedures (2.5% vs 2.3%).29 In addition, there were no differences in total pregnancy loss between transabdominal CVS and amniocentesis in the second trimester (6.3% vs 7%, relative risk [RR], 0.90 [95% CI, 0.66-1.23]).29 Transcervical CVS was analyzed in a retrospective series by Donner et al.30 The group reported a fetal loss rate of 2.5% in 1251 procedures. However, a meta-analysis by Alfirevic et al32 showed that, compared with second-trimester amniocentesis, transcervical CVS probably carries a higher risk of total pregnancy loss and spontaneous abortion, although exact risks could not accurately be obtained

Currently, the majority of patients undergoing an invasive procedure are a priori at a higher risk of pregnancy loss, due to the indication to test itself—ie, elevated nuchal translucency, low serum pregnancy-associated plasma protein A (PAPP-A) levels, fetal malformations, and intrauterine growth restriction (IUGR).

Vaginal Bleeding

The frequency of vaginal bleeding is around 10%. It seems to be more frequent in the transcervical approach (up to 30% of cases). The presence of light vaginal bleeding does not necessarily increase the risk of pregnancy loss. Therefore, whenever a patient experiences a light vaginal bleeding after a procedure, she can be reassured that most of the time there will be no pregnancy loss.14,24

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Jun 19, 2022 | Posted by in OBSTETRICS | Comments Off on Invasive Diagnostic Procedures

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