Learning Objectives:After participating in this continuing professional development activity, the provider should be better able to:
1. Distinguish among the various types of cytogenetic abnormalities, copy-number variants, and single-gene disorders in relation to prenatal diagnostic testing.
2. Choose appropriate cytogenetic and molecular genetic tests for prenatal diagnosis.
3. Outline patient counseling on the risks, benefits, and limitations of prenatal diagnostic tools and explain when to refer appropriately for genetic counseling.
Over the past 15 years, significant advancements in molecular genetics have expanded the scope and depth of prenatal diagnostic testing that can be performed during pregnancy. With modern sequencing technology and increased uptake of noninvasive screening for common aneuploidies, there has been a decrease in the use of diagnostic testing during pregnancy.1,2 In addition to improved screening, with more informed and shared decision models of care, there has been a lower uptake of prenatal diagnostic testing.3 Additionally, over the past few decades the indications for diagnostic testing have changed from determining common aneuploidies such as Down syndrome for individuals with advanced maternal age to determining a much broader range of genetic conditions for all pregnant persons, with the most common indication being ultrasound abnormalities of the fetus, or parents being known carriers for a condition.2,4 The American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal Fetal Medicine (SMFM) recommend that all pregnant persons, regardless of age or risk, be counseled about screening and diagnostic testing options for aneuploidy and other genetic conditions during each pregnancy.5
Prenatal care practitioners should be prepared to educate and counsel pregnant patients on the fundamental genetic screening and diagnostic testing options to empower them to make testing decisions in line with their own goals and values. Prenatal diagnosis is performed for many reasons, and it is important that patients understand that diagnosis is not solely performed for assistance in decision-making regarding pregnancy termination. Prenatal genetic counselors are a vital resource for providers and patients to discuss more in-depth pre- and posttest counseling and the next steps after high-risk screening results. They also assist with counseling and testing options after discovery of abnormal findings on a prenatal ultrasound. However, there is a critical shortage of prenatal genetic counselors in the United States,6 and therefore a large amount of this counseling may be performed by prenatal care providers themselves. Therefore, it is important for prenatal practitioners to have a foundational understanding of prenatal diagnostic testing methods and genetic analysis to provide patient-centered pre- and posttest counseling for appropriate testing options, interpretation of results, recognition of when to seek genetic expertise for counseling, and when to make modifications of medical care plans in response to genetic testing results.
This review article focuses on prenatal diagnosis methods and genetic diagnostic testing; this review does not discuss prenatal genetic screening. The diagnostic tests that have been discussed include preimplantation genetic diagnosis, chorionic villus sampling (CVS), amniocentesis, and cordocentesis. With samples from embryo, trophoectoderm, placental villi, or fetal cells, there are various genetic tests that can be performed including karyotype, microarray, next-generation sequencing for single-gene conditions, and whole exome/genome sequencing. This article discusses the advantages and limitations of each procedure for prenatal diagnosis and for each genetic testing methodology.
Indications for Diagnostic Testing
Our understanding of genetics and genomics has significantly advanced over the past few decades; however, we still have an incomplete understanding regarding inheritance and complexity of many genetic conditions. Prenatal diagnosis is possible for many but not all conditions. Even after confirmation of a genetic diagnosis with diagnostic testing, it is important for the patient to understand that in most cases this does not predict details of the outcome and severity of the condition. Chromosomal abnormalities, copy-number variant conditions (microdeletions/microduplications), and single-gene conditions are generally able to be detected through analysis of placental or fetal tissue.
There are various indications for prenatal diagnostic testing, and these include advanced maternal or paternal age, abnormal results from aneuploidy screening, fetal structural anomalies on ultrasound, parental translocation carrier, parental carrier of genetic condition, previous fetus or child with aneuploidy or a genetic condition, and maternal request.4 Regardless of risk factors, all pregnant persons should be counseled about screening and diagnostic testing options for aneuploidy and other genetic conditions during each pregnancy.5
To assist an individual in their decisions regarding screening versus diagnostic testing, providers can evaluate the patient's risk factors for genetic conditions in their offspring by detailed family history, prior obstetric history, calculation of aneuploidy risk based on maternal age or genetic screening results, and a person's ethnic background. However, there are known limitations in reliability of ethnicity data and for this reason some providers choose population-based expanded carrier testing.7,8 Genetic counselors are a valuable resource to additionally assist with risk assessment and help patients to make decisions that are in line with their beliefs, values, and traditions.
The Basis of Genetic Conditions
Before a discussion of different genetic diagnostic testing options, it is important to first review a basic framework of inheritance and genetic disease. Human DNA is mostly packaged into 46 nuclear chromosomes, including 44 autosomes and 2 sex chromosomes. Nuclear chromosomes have primary, secondary, and tertiary structure; genetic conditions can arise due to errors or variation at any level of this structure.9 Primary structure is the sequence of nucleotides that make up DNA; differences in nucleotide sequence can result in normal human variation or genetic disease. Secondary structure describes 2 complementary strands of DNA held together by hydrogen bonds to form a double helix and, these strands can separate to allow recombination that can introduce normal variation or genetic disease. Tertiary structure describes DNA interaction with histone proteins, where DNA is wrapped around histones like thread on a spool. Based on molecular changes of histone proteins, this can alter transcriptional activity of certain regions of the DNA and these epigenetic changes can introduce variation or genetic disease.9
To form gametes for reproduction, diploid (2n) primordial germ cells with 46 chromosomes undergo 2 phases of meiosis to form haploid (1n) gametes with 23 chromosomes. During this process homologous recombination occurs via crossing over, and genetic information from homologous chromosomes is exchanged. In females, oogenesis begins in utero and is paused in prophase I. Beginning with puberty, one or a few gametes resume meiosis each ovulatory cycle. At the point of ovulation, meiosis I, including homologous recombination, has been completed. Meiosis II is only completed if and when fertilization occurs. In males, spermatogenesis begins at puberty and continues throughout reproductive life.9
Chromosomal abnormalities can result from abnormal number or structure of one or more chromosomes.10 The most common chromosomal abnormality is aneuploidy, which is the presence of an extra or missing chromosome or chromosomes. It is believed that meiotic nondisjunction, usually during meiosis I, is the primary cause of aneuploidy. In nondisjunction events, a pair of chromosomes fails to separate properly. This results in a gamete, which should be haploid for 23 chromosomes, being diploid or lacking copies of a chromosome.10 There are 3 nonmosaic autosomal aneuploidy conditions that are viable prenatally: trisomy 21, trisomy 18, and trisomy 13. There can also be aneuploidies of the sex chromosomes. It is also possible to have one or more extra sets of all the chromosomes, namely triploidy (3N) or tetraploidy (4N).
In addition to aberrations in chromosome number, there can be structural rearrangements of chromosomes including deletion/duplications, translocations, and other rearrangements (Figure 1). Deletion and duplication syndromes occur when there are missing or extra copies of a chromosome segment. These copy-number variants result from unrepaired chromosome breakage during cell replication. There are certain hotspots in the genome that are prone to breakage, resulting in a higher incidence of certain deletion or duplication conditions than would be expected by chance.10 A copy-number variant condition (deletion or duplication syndrome) can arise de novo as a new event in the offspring or can be passed through generations from parent to child.11 The size of copy-number variants can differ, and when they are larger than 5 to 10 Mb they can be identified via cytogenetic analysis like karyotype. Microdeletions and microduplications, smaller than 5 to 10 Mb, are often detected via molecular methods like chromosomal microarray.12 Some common deletion syndromes are Cri du chat syndrome (5p-), DiGeorge (22q11.2) Prader-Willi and Angelman syndrome (15q11-q13), and Williams syndrome (7q11.23).11
Chromosomal inversions occur when one chromosome has broken apart at 2 locations and is then repaired with the intermediate segment inverted in direction (Figure 1). Because inversions do not result in the loss of genetic material (balanced rearrangement), they are often of no clinical significance. However, individuals with an inversion may be more likely to produce gametes with chromosomal abnormalities due to challenges in chromosome alignment during meiosis I. If the inversion does not span the centromere (paracentric inversion), viable gametes are rarely produced. However, when the translocation spans the centromere (pericentric inversion), viable gametes may be produced and the incidence of producing offspring with an unbalanced karyotype is approximately 5% to 10%.10
Ring chromosomes form when a chromosome undergoes 2 breaks, and the broken ends are joined into a circular structure (Figure 1). These ring chromosomes are unstable and have difficulty aligning during cell division. As a result, daughter cells may have partial or complete aneuploidy.10 Ring formation has been observed for all human chromosomes. The chromosome involved and extent of crucial gene deletion are the largest factors impacting phenotype of patients with a ring chromosome.12 Ring chromosomes can often be detected using karyotype. Fluorescence in situ hybridization (FISH) and microarray can then be used to further elucidate the specific genomic breakpoints involved.12
Isochromosomes form when one arm of a chromosome is lost and replaced by a mirror image of the other arm (Figure 1). A person with 46 chromosomes including 1 isochromosome will have partial monosomy for the lost chromosome arm and partial trisomy for the duplicated chromosome arm. The most common isochromosome is formed by loss of the short arm of the X chromosome and duplication of the long arm. Patients with this X isochromosome genetic abnormality often present with Turner syndrome.10
Translocations occur when there is an exchange of genetic segments between 2 chromosomes, and these exchanges can be reciprocal or nonreciprocal (Figure 1). Reciprocal translocations are 2-way transactions, transferring genetic material between 2 nonhomologous chromosomes, often resulting in a balanced genome. Because very little to no genetic material is lost, balanced reciprocal translocations are often without phenotypic effect. However, balanced reciprocal translocations are associated with a high risk of producing genetically unbalanced gametes and resultant zygotes with partial monosomy or trisomy.
Reciprocal translocations should be considered in couples after 2 or more spontaneous abortions and in males with infertility.10 Nonreciprocal translocations occur when genetic information is transferred from one chromosome onto another nonhomologous chromosome without further reciprocal exchange. Robertsonian translocations occur when 2 acrocentric chromosomes fuse at the centromere, resulting in the loss of both chromosome short arms. Individuals with Robertsonian translocations have 45 chromosomes and have partial monosomy for the short arms of both chromosomes involved (Figure 1). Despite this loss of genetic information, patients often present with a normal phenotype as the short arms of acrosomal chromosomes have very low gene density.10 These individuals are, however, at an increased risk of creating unbalanced gametes and resultant unbalanced zygotes. Inheritance of a Robertsonian translocation involving chromosome 21 accounts for about 4% of Down syndrome cases. For parents with these translations, the Down syndrome recurrence is higher than it is among the general population.10
Insertions, another type of nonreciprocal translocation, occur when a segment is removed from one chromosome and placed into a different chromosome inverted relative to the centromere (Figure 1). Although patients with insertions are often phenotypically normal, the incidence of producing a child with partial aneuploidy can be as high as 50%.11
Concept of Mosaicism in Relation to Prenatal Diagnosis
Abnormalities in chromosome number can be mosaic, meaning the abnormal number of chromosomes is not present in all of the cells (Figure 2). Mosaicism occurs when two or more groups of cells within a tissue or individual differ genetically but are derived from a single zygote. The extent of mosaicism, or how many cells are impacted, and the point in development during which the mutation occurred are both major determinants of clinical presentation and outcome.10 Mosaicism is identified in approximately 0.1% to 0.4% of CVS and 0.25% of amniocentesis samples. With CVS, this may represent a mosaic event in the placenta and be confined placental mosaicism in 1% of cases (Figure 2).10,13 Counseling patients regarding the implications of chromosomal mosaicism can be complex, and referral to a genetic counselor or expert in genetics may be useful.13
Prenatal Diagnostic Tools
The methods to obtain placental or fetal samples for genetic testing include preimplantation genetic diagnosis, CVS, amniocentesis, and percutaneous blood sampling. Determination of the optimal method to obtain a sample for genetic testing depends on the timing in pregnancy, type of genetic testing desired, and indication for genetic testing (Figure 3). Each of these procedures has advantages and limitations (Table 1).
Preimplantation Genetic Diagnosis: Embryo Biopsy
Patients who plan to undergo in vitro fertilization have an opportunity to evaluate embryos with genetic testing before implantation, allowing for selection of embryos without aneuploidy or an underlying genetic condition for transfer to the uterus.10,14 In the past, preimplantation genetic testing was performed on the polar body of a prefertilized oocyte or a single blastomere from cleavage-stage embryos, typically on day 3 of culture. However, results were less than optimal, and overall pregnancy rates of embryo transfer were adversely affected. At present, the most common methodology is biopsy of approximately 5 to 10 cells from the trophectoderm (which becomes the placenta), at the blastocyst stage-usually 5 to 6 days after retrieval.10,14 Because of possible mosaicism, the preimplantation genetic testing results from trophectoderm biopsy may not accurately reflect genetics of the fetus (inner cell mass of the embryo). There may be false-positive or false-negative results with preimplantation genetic diagnosis, and for this reason, prenatal diagnostic testing during pregnancy via CVS or amniocentesis should be offered to all patients who have become pregnant after preimplantation genetic diagnosis.10,14 The diagnostic error rates for both preimplantation genetic testing-aneuploidy (PGT-A) and -monogenic (PGT-M) are approximately 1%.30
There are two main types of preimplantation genetic testing: PGT-A to screen embryos for aneuploidy and PGT-M to assess for a single-gene condition in the embryo.14 With each of these genetic tests, a biopsy is taken from the embryonic trophectoderm and the embryos are then frozen until the results are available. Once the genetic testing has been finalized, the physician can help the patient select an embryo or embryos without aneuploidy or a single-gene disorder. In essentially all cases of PGT-M, embryos are also tested for aneuploidy, and only one euploid embryo is transferred at a time. For assessment of single-gene conditions, the specific pathogenic variant in the family or parent must be known before genetic testing in the embryo. The genetic testing employed depends on the desired results; for PGT-A, typically chromosomal microarray is used and for PGT-M, next-generation sequencing is used for monogenic single-gene conditions.10,14
Chorionic Villus Sampling
CVS for prenatal diagnosis obtains a tissue sample from the placenta and is performed between 10 and 13 weeks' gestation with a processing time of 5 to 7 days.13 CVS can be performed via a transcervical or transabdominal approach under ultrasound guidance and avoids entry into the gestational sac.13 There appears to be no significant difference in the safety profile between 2 the different approaches, and the location and accessibility of the placenta determines the best strategy.5,13,15,16 The major benefit of CVS over amniocentesis is early provision of test results, increasing reproductive options regarding the pregnancy.
Both methods of transcervical and transabdominal CVS begin with an ultrasound to confirm fetal size, fetal heart activity, and orientation of the placenta. With transcervical CVS, the placental location and potential path for the catheter is first verified on transabdominal ultrasound. The patient is then positioned in dorsal lithotomy, and the vulva/vagina/cervix are prepped with a povidone-iodine solution. The catheter is then passed through the cervix and into the placenta under ultrasound guidance. A syringe with cell culture media is then attached to the proximal end of the catheter and negative pressure is applied as the catheter is slowly removed. After the procedure, villi can often be seen by the naked eye or detected with low-power microscopy. The procedure can be repeated a second time if too few villi are retrieved with the first pass.9 With transabdominal CVS, a 19- or 20-gauge needle is placed through the abdomen under ultrasound guidance into the long axis of the placenta, and villi are aspirated with a syringe filled with cell culture media as the needle moves back and forth through a section of the placenta. Although transcervical CVS is ideally performed before 14 weeks' gestation, transabdominal CVS can be performed throughout pregnancy. For example, with anhydramnios or oligohydramnios, transabdominal CVS is often feasible throughout pregnancy.9
Many studies have been conducted to compare the safety profile of CVS to second-trimester amniocentesis. A 2015 meta-analysis of 46,287 patients demonstrated a CVS procedure-related loss rate of 0.22%.17 Although CVS and amniocentesis on aggregate are equally safe, studies have shown that CVS often has a longer learning curve than amniocentesis, and that operator experience impacts the incidence of pregnancy loss.9,18 In procedures performed before week 10 of gestation, the incidence of limb-reduction defects is very low. A 1994 analysis by the World Health Organization demonstrated that the incidence of limb reduction defects did not differ between pregnancies that underwent CVS compared with the general population.19 Other complications of CVS include vaginal spotting or bleeding in up to 32% of transcervical procedures, and fluid leakage and infection, both less than 0.5%.13
Chorionic villi originate from the trophoblast, the extraembryonic portion of the blastocyst.10 Because CVS is examining extraembryonic tissue, mosaicism found in the cultured sample may or may not reflect fetal mosaicism, resulting in ambiguous results for 1% of CVS samples (Figure 2). With the suspicion of confined placental mosaicism from CVS results, confirmation by amniocentesis is advised.10,14 Another limitation of CVS is its inability to evaluate for genetic conditions such as imprinting disorders where methylation testing is indicated. The placenta has a methylation profile distinct from the embryo's methylation profile making amniocentesis the preferred test for these examinations.
Amniocentesis
Amniocentesis for prenatal diagnosis is generally performed between 15 and 20 weeks' gestation, but can be performed at a later gestational age, and has a processing time of 7 to 14 days.10,13 Fetal cells are present in the amniotic fluid and these cells can be sent for cytogenetic and molecular genetic testing.
The procedure begins with ultrasound to determine viability, fetal lie, placental location, cord insertion, and identification of a fluid pocket free of umbilical cord and fetal parts. The abdomen is typically cleaned with povidone-iodine solution and with sterile technique a 22-gauge spinal needle is inserted into the fluid pocket under ultrasound guidance. To avoid contamination with maternal tissue, the first 2 mL of aspirated fluid is discarded and then an additional 20 to 30 mL of fluid is collected. A second attempt in another location can be performed if the first attempt is not successful.9 This fluid can additionally be sent to rule out spina bifida by analysis of [alpha]-fetoprotein or acetylcholinesterase, for viral studies if there is a concern for congenital infection, or for biochemical testing. Transplacental passage is typically avoided if possible; however, if unavoidable the procedure-related loss rate is not different with nontransplacental versus placental approach.20 Early amniocentesis, performed between 10 and 13 weeks, is not recommended, as this has concern for disruption, amniotic bands, and higher rates of complications including pregnancy loss.13
The most significant risk associated with amniocentesis is pregnancy loss. A recent meta-analysis of miscarriage risk after amniocentesis estimated the fetal loss rate of 0.11% (1 in 900).17 With experienced providers, the rate of loss after amniocentesis is estimated at 0.1% to 0.3%. Another risk with amniocentesis is amniotic fluid rupture, with a relative risk of 4.25%. This can resolve in many cases and most commonly results in good pregnancy outcomes.21-23 Other risks associated with amniocentesis include a small risk of bleeding, infection, and failure of cell culture in 0.1% of cases.13 After the procedure, individuals typically have lower abdominal discomfort that resolves in 24 to 48 hours.
Percutaneous Umbilical Blood Sampling (Cordocentesis)
Percutaneous umbilical cord sampling (PUBS) is primarily performed when there is concern for fetal anemia. From this fetal blood sampling, cytogenetic and molecular genetic testing on the fetus can also be performed. The risk of fetal loss after PUBS appears to be 2.5%; however, the risk of fetal loss is also dependent on whether the fetus had other complications including hydrops before the procedure.24
Fetal blood can be obtained from either the cord or the fetus. Cord blood is easiest to obtain at the site of placental insertion whereas the hepatic vein is the most accessible source within the fetus. To verify that fetal, as opposed to maternal, blood was obtained, a mean corpuscular volume analysis can be performed. An alternative method involves injecting sterile saline at the site of collection, followed by observation of microbubble movement.9
Genetic Tests Performed After Prenatal Diagnosis Procedures
The cytogenetic or molecular test performed on diagnostic samples depends on the indication for the test and the type of sample available (Table 2). Cytogenetic analysis is performed with FISH and karyotype. Chromosomal microarray is warranted if fetal anomalies are identified on ultrasound to evaluate for microdeletions/microduplications. With a specific anomaly or phenotype or genetic syndrome, after normal chromosomal microarray results, next-generation sequencing panels or whole exome/genome sequencing may be performed; however, this is not yet standard of care.
Karyotype
Karyotype is a cytogenetic analysis where condensed chromosomes are visualized after short-term culture of fetal cells. Each chromosome has a unique banding pattern created by varying regions of euchromatin and heterochromatin. The pattern of the bands is used to identify chromosomal abnormalities. This technique can be used to identify aneuploidies and structural chromosomal rearrangements larger than 5 to 10 Mb.13 It is additionally able to detect balanced chromosomal rearrangements.
Fluorescence In Situ Hybridization
FISH is a rapid method for detection of common aneuploidies and can be performed on direct specimens or cultured cells. The results may be less accurate than other tests, and thus FISH is always followed by confirmatory karyotypic analysis. Chromosomes are fixed onto a microscope slide and then bathed in fluorescent-labeled DNA probes specific for the desired region of chromosomes. The probe then hybridizes to complementary DNA regions and can be visualized using a fluorescence microscope. Confirmatory testing of abnormal direct FISH results should be performed by karyotype or chromosomal microarray.11
Chromosomal Microarray Analysis
Chromosomal microarray analysis (CMA) yields higher-resolution results to identify microdeletions and duplications throughout the human genome. The risk for microdeletions and duplications is not associated with increasing maternal age. Deleted or duplicated sections of DNA of at least 1000 base pairs in size that differ from reference genome are known as copy-number variants (CNVs). Copy-number variants can have clinical significance and result in disease phenotype (pathogenic variant), have no clinical significance (benign), or the actual clinical significance can be unknown (variant of unknown significance, ie, VUS).25 CMA can additionally identify aneuploidy and unbalanced chromosomal structural rearrangements; however, it cannot determine balanced chromosomal rearrangements, locations of chromosomal rearrangements, or cases of low-level mosaicism.
Microarray analysis is the first line of genetic testing for fetal structural anomalies on ultrasound. CMA has been shown to identify an additional 6% of genetic conditions in fetuses with ultrasound anomalies and normal karyotype.26 However, there is an exception for cases where common aneuploidy is highly suspected. In these instances, karyotype is the preferred method for analysis.13,25 In addition, CMA is the recommended test for products of conception and stillbirth because it does not require actively dividing cells for analysis, as does karyotyping.
Next-Generation Sequencing
Although microarray does identify more genetic abnormalities than traditional karyotype, most fetuses with anomalies have normal microarray findings. In these cases, next-generation sequencing may illuminate the genetic basis of disease.25 There are next-generation sequencing panels that may be performed if there is suspicion for a specific genetic condition or phenotype such as Noonan syndrome or skeletal dysplasia panels.
A more comprehensive approach involves whole exome sequencing (WES) or whole genome sequencing (WGS). WES analyzes sequences from coding DNA or exomes versus WGS, which involves analysis of the entire genome. With WES in prenatal cases, trio sequencing is the typical approach. Trio sequencing involves obtaining samples from both biological parents to compare with fetal information. This increases the diagnostic yield by filtering out thousands of uninformative genomic variants. With limited data, WES has been shown to detect a genetic condition in 20 to 30% of anomalous fetuses with normal karyotype and microarray.27 The American College of Medical Genetics and Genomics recommends consideration of WES when standard methods, microarray and karyotype, have failed to determine a diagnosis and there are multiple congenital anomalies present.28 ACOG recommends that if WES is being considered for a prenatal diagnosis, it is vital to involve an individual with expertise in clinical genetics in the pre- and posttest counseling, discuss challenges of longer turnaround time of this test compared with other genetic testing analysis, and discuss concern for other incidental findings for pathogenic variants in the parents with this testing.29
Some of the challenges with WES for prenatal genetic diagnosis include longer turnaround times, insurance not covering the cost of testing, and discovery of clinically relevant but incidental findings in a parent or fetus. Both the ACOG and the SMFM do not recommend WES as a routine prenatal diagnostic test; however, WES can be considered for a narrow set of specific clinical indications or limited to research basis at this time.
Conclusion
Technological advancements in molecular genetics have drastically changed the landscape for prenatal diagnostic testing performed preimplantation or during pregnancy. The ACOG and the SMFM recommend that all pregnant persons, regardless of age or risk, be counseled about screening and diagnostic testing options for aneuploidy and other genetic conditions during each pregnancy. Gestational age, reasons for testing, and specific genetic analysis all contribute to the optimal method to obtain a placental or fetal sample. There is a small risk for fetal loss with most methods of prenatal diagnosis. These risks versus the benefits of obtaining information need to be conveyed to the patient. It is the task of prenatal providers, with the assistance of genetic counselors, to give patients information to make decisions in line with their own values and beliefs. The ultimate decision of whether or not to pursue prenatal diagnosis testing is determined by the patient.
REFERENCES