CLINICAL OBSTETRICS AND GYNECOLOGY Volume 57, Number 1, 210–225 r 2014, Lippincott Williams & Wilkins
Noninvasive Prenatal Detection of Aneuploidy CHRISTOPHER ROBINSON, MD,* DIRK VAN DEN BOOM, PhD,w and ALLAN T. BOMBARD, MDw z *Department of Obstetrics and Gynecology/Division of MaternalFetal Medicine, University of Virginia, Charlottesville, Virginia; w Sequenom Inc.; and z Department of Obstetrics and Gynecology/ Division of Reproductive Medicine, University of California San Diego, San Diego, California Abstract: Noninvasive prenatal testing (NIPT) uses cell-free fetal DNA from the plasma of pregnant women to provide valuable information about the potential risks for fetal aneuploidy. This article provides a historical overview of both invasive diagnostic testing and serum screening approaches, both biochemical and the newer molecular noninvasive prenatal testing assays, used to identify patients who would be best served by invasive testing. Key words: noninvasive prenatal testing, NIPT
which remains the gold-standard for prenatal aneuploidy diagnosis at the time of this article. Understanding the historical advances in prenatal aneuploidy detection alongside advances in biomolecular science and bioinformatics in the past decade, can provide the context for a full understanding of the role and potential for use of new, molecular approaches to noninvasive methodologies in the prenatal detection of aneuploidy, known currently as noninvasive prenatal testing (NIPT)—and the subject of this chapter.
Prenatal aneuploidy detection and screening has been an area where major advances in technology have led to improved detection and accuracy over the past few decades. Recently, advances in genomic sequencing and bioinformatics have led to aneuploidy detection techniques that are approaching the detection rate and accuracy seen in amniocentesis and chorionic villus sampling-derived karyotype analysis,
Detection of Fetal Aneuploidy: A Brief Overview of Screening and Diagnosis Historically, prenatal diagnosis began to expand dramatically with growing interest in pregnancies carrying a fetus with Down syndrome. Down syndrome is a genetic disorder caused by the presence of all or part of an extra 21st chromosome
Correspondence: Allan Bombard, MD, Sequenom Inc., San Diego, CA. E-mail: [email protected] D.V.D.B. and A.T.B. are full-time employees of Sequenom, Inc. The other author has no conflict of interest to declare. CLINICAL OBSTETRICS AND GYNECOLOGY
210 | www.clinicalobgyn.com
/
VOLUME 57
/
NUMBER 1
/
MARCH 2014
Noninvasive Prenatal Detection of Aneuploidy (trisomy 21 or T21). It is named after John Langdon Down, the British physician who described the clinical features in 1866. The clinical disorder was identified as most commonly being due to an extra copy of chromosome 21 (trisomy) by Lejeune in 1959, and is characterized by a combination of major and minor physical features. Often, Down syndrome is associated with some impairment of cognitive ability and physical growth as well as hallmark physical features. Down syndrome can be identified during pregnancy or at birth. Trisomy 21 is the most common chromosomal aneuploidy in live born infants. The overall incidence is approximately 1 in 800 births in the general population but this risk increases to 1 in 35 term births for women aged 45 years. Advanced maternal age is only one factor contributing to the increased risk. When other factors, such as positive serum biochemical screening results or fetal ultrasound indicative of aneuploidy or previous or family history of aneuploidy are included, the incidence can be as high as 1 in 15 live born infants. The presence of any one of these risk factors places the pregnancy in the category of high risk for Down syndrome. Prenatal fetal assessment and testing can be provided through both invasive and noninvasive methodologies. Before the development of serum biochemical screening tests in the 1980s, aneuploidy was detected exclusively through invasive technology using genetic amniocentesis or chorionic villus sampling (CVS) to provide a sample of the fetal genotype for analysis.1 Invasive testing, which began almost 5 decades ago consists of CVS, which can be performed between 9.5 and 12.5 weeks’ gestation, or genetic amniocentesis that can be performed most commonly after 14 weeks’ gestational age.2 Analyses on specimens obtained by these invasive tests, owing to superior sensitivity and low false-positive rates, are considered
211
diagnostic and therefore the gold-standard in prenatal diagnosis. These surgical procedures preceded the availability of noninvasive assessment of fetal risk for certain heritable disorders. However, due to a strong desire on the part of patients and their physicians, less risky, noninvasive approaches evolved. Noninvasive screening approaches involve the use of risk factors for fetal aneuploidy. The first such screening tests were advanced maternal age and prior history—also the first indications for invasive diagnostic testing. Subsequently, with the recognition that abnormalities in the levels of certain biochemical markers could provide better identification of those women at an increased risk, the introduction of serum markers was soon followed by advances in fetal ultrasound that could also substantially aid in the identification of pregnancies at risk.1 The identification and quantification of specific, indirect fetal biomarkers found in the maternal serum presaged the advent of novel, new target assays in the maternal plasma that now constitute the latest advances in NIPT. When present in concentrations outside of the normal ranges for varying gestational ages, pregnant women who manifest positive serum biochemical screening tests were identified as patients who might be at an increased risk for fetal disease and who would derive the greatest benefit from invasive diagnostic testing, despite the risks of such procedures. Although these serum biochemical screening tests offer 70% to 90% detection, they are compromised by a 2.5% to 5% screen (usually false)-positive rate. Before the advent of NIPT there existed 4 principle risk factors for fetal aneuploidy: personal or family history; advanced maternal age; target serum analytes, biomarkers associated with aneuploidy; and ultrasound findings indicative of structural anomalies. A patient who has been identified as being at an www.clinicalobgyn.com
212
Robinson et al
increased risk for fetal aneuploidy because of the presence of one or more of these risks was traditionally felt to be a candidate for invasive prenatal diagnosis. This changed with the ACOG Committee Opinion #77, issued in January 2007 and which, for the first time, suggested that given adequate informed consent, any patient could be offered invasive diagnostic testing.3 Since then, the standard-ofcare has been that after counseling and the granting of informed consent, patients found to be at an increased risk may opt a priori for invasive diagnostic testing that provides fetal tissue diagnosis with a very high sensitivity and low false-positive rates. This approach is far more definitive than the alternative noninvasive prenatal serum biochemical screening methodologies that have a lower detection and higher false-positive rates for aneuploidy. Unlike invasive procedures, which carry a small but definite risk for fetal and maternal complications, noninvasive serum biochemical testing approaches have no such risks—but have poorer predictive values. Patients therefore are presently urged to consider the risks and benefits of both such approaches—noninvasive serum biochemical screening tests, with relatively poor sensitivity and specificity but little risk of fetal loss, and invasive diagnostic testing, with excellent sensitivity and specificity but the small but real risk for both fetal loss and maternal complications. The value proposition presented by NIPT is the ability to dramatically improve the sensitivity and specificity of risk assessment by noninvasive approaches to a level afforded by invasive testing, whereas retaining the safety of simple phlebotomy and avoiding risks to mother and baby in the vast majority of the patients.
Invasive Prenatal Diagnosis Invasive diagnostic sampling includes CVS in the late first trimester, genetic amniocentesis in the early second www.clinicalobgyn.com
trimester, or, less commonly, percutaneous umbilical blood sampling (PUBS) in the mid second trimester. CVS, genetic amniocentesis, and PUBS are invasive procedures, in that each involves inserting instruments into the uterus, and therefore add a small risk to the pregnancy of causing fetal injury or miscarriage. All 3 sampling methods are followed by karyotyping with or without fluorescence in situ hybridization (FISH), microarray and/or quantitative fluorescent polymerase chain reaction (QF-PCR), which includes amplification, detection, and analysis of chromosome-specific DNA sequences. The analyses from specimens obtained by these invasive diagnostic procedures are presently the only way to confirm a diagnosis of Down syndrome, or other abnormalities, and are usually offered to families who either screen positive by serum biochemical means or may have an increased chance of having a child with a chromosome or other heritable abnormality that is amenable to laboratory diagnosis. Noninvasive prenatal diagnostic processes have, as a result, been a longstanding research theme in prenatal medicine. In January 2007, the American Congress of Obstetricians and Gynecologists issued an updated Practice Bulletin that recommends that all pregnant women be offered both screening and the option of invasive diagnostic testing for fetal trisomy 21.4 It is well appreciated that the prevalence of fetal chromosomal aneuploidy varies as a function of both maternal and gestational age.5 For example, the prevalence of fetal chromosomal abnormalities exceeds 50% in first trimester, noted in spontaneous abortions.6 By contrast, at term, fetal aneuploidy can be found in 6% to 13% of stillbirths.7 Chromosomal abnormalities that are compatible with extrauterine life, but with significant morbidity and mortality, occur in B0.6% of newborns.8 Thus, invasive procedures—CVS, genetic amniocentesis, and PUBS—are surgical
Noninvasive Prenatal Detection of Aneuploidy procedures used to obtain fetal or fetally derived tissues for the detection of chromosomal abnormalities and have been key in enabling the assessment of the prenatal, intrauterine fetal condition and state. Although CVS and amniocentesis are common first and second trimester, respectively, surgical procedures, cordocentesis, provides a primary advantage of rapid karyotype analysis within 24 to 48 hours after procedure. However, it is rarely utilized owing to higher pregnancy loss rates. Alternative methods for rapid detection of common aneuploidies by FISH and QF-PCR are now routinely used in the United States and Europe, facilitating a more rapid result for patients undergoing CVS and amniocentesis. As mentioned above, the risk for fetal loss associated with these invasive procedures, driving the search for noninvasive alternatives with better performance than serum biochemical screening tests. Amniocentesis was the first invasive procedure developed to evaluate the fetal condition—whether metabolic or genetic. Amniocentesis has been reported as early as 1877 when Prochownick, Von Schatz, and Lambi performed the procedure in the third trimester of pregnancy. A single report from 1919 described the removal of amniotic fluid due to hydramnios in a third trimester pregnancy.9 Bevis10 in 1953 used amniotic fluid obtained by amniocentesis every 2 weeks to assist in the management and prediction of severity of Rh alloimmunization. The initial use of amniocentesis in the detection of genetic disease was described in 1956 in the journal Nature.2 Fuchs and Riis reported the ability to detect fetal sex based on the presence or the absence of the Barr body among cells obtained at amniocentesis. The ability to determine fetal sex by amniocentesis would lead to the use of invasive testing for the antenatal detection of sex-linked hereditary disorders such as Hemophilia A (1960) and Duchenne muscular dystrophy (1964).11
213
Nadler and Gerbie went on to publish the landmark article entitled ‘‘Role of amniocentesis in the intrauterine detection of genetic disorders’’ in the New England Journal of Medicine in 1970 where they described 162 amniocentesis procedures with a 97% success rate for chromosome analysis with a single amniocentesis procedure.12 This documented experience would form the basis for the utilization of invasive prenatal detection of the fetal genetic state in the second trimester. Around the same time, CVS was shown to be an alternative mechanism for assessment of the fetal state.13,14 The initial attempts at CVS were performed in 1968 with a rigid 5-mm endoscope with direct visualization of the chorion for direct biopsy and retrieval of villi. This initial approach in 1968 by Mohr in Scandinavia was successful in over 96% of the attempts but was complicated by unacceptable rates of bleeding, infection, and failed cultures.15 As a result, this methodology for CVS was abandoned as amniocentesis was less complicated and had a higher success rate for completed karyotype analysis. Ward et al16 would use ultrasound guidance with the passage of a transcervical catheter in 1983 to successfully biopsy the chorionic plate with 89% success. This methodology would be further refined into the modern CVS procedure for prenatal diagnosis. When considering prenatal diagnosis, there are many potential indications for performing amniocentesis or CVS in providing information concerning the fetal intrauterine state. Patients may desire testing for aneuploidy, single-gene defects, neural tube defects, or for confirmation of fetal blood type in cases of possible alloimmunization risk. When considering the totality of cases undergoing prenatal diagnostic procedures with amniocentesis or CVS, the majority of the procedures (>90%) are performed for aneuploidy detection among women who desire this information or who www.clinicalobgyn.com
214
Robinson et al
are deemed at high risk for aneuploidy. The ACOG supports prenatal diagnostic intervention availability for all women who desire this testing regardless of perceived/ actual risk or maternal age.4 An important aspect of prenatal diagnostic testing involves the pretest counseling regarding the risks and benefits of amniocentesis and CVS. CVS has the advantages of offering first trimester prenatal diagnostic karyotype information and can be performed directly and provide rapid results in 2 to 4 hours after procedure with direct preparation or after tissue culture for 48 to 72 hours with results within a week. There is a 99.7% rate of accuracy of cytogenetic results with CVS obtained chorionic villus material among more than 6000 patients enrolled in the United States Collaborative Study. This group found that a repeat procedure was necessary in 1.1% of patients due to maternal cell contamination or mosaicism noted in the culture.17 As CVS involves biopsy of the chorionic plate, contamination of the biopsy material can include maternal decidual tissue that can lead to erroneous results. This can be minimized through ensuring adequate villus tissue in the specimen and maternal cell contamination analysis to examine for polymorphisms that would allow distinction between maternal and fetal cells. Mosaicism also complicates around 1% of CVS specimens.17,18 When this is found, consideration of whether this is due to culture, confined placental mosaicism, or representative of the actual fetal state must be considered. To confirm this, a repeat procedure is often needed that is most commonly an amniocentesis. Amniocentesis has the benefit of avoiding the higher potential for maternal cell contamination and culture-based mosaicism seen in CVS, but carries the disadvantage of being performed in the second trimester (generally 15 to 20 wk’) that would not allow for early pregnancy termination if desired with an abnormal result. www.clinicalobgyn.com
CVS and amniocentesis have been examined with regard to fetal loss rates associated with each procedure. The overall rate of prenatal loss with a CVS procedure is greater than that of amniocentesis, though, this is most likely due to the increased background pregnancy loss rates seen at 6 to 16 weeks gestational age.19 Procedure-related loss rates for amniocentesis have been reported to be between 1/300 and 1/500 in the literature with even lower risks in more contemporary reports.20,21 A large, national registry describing an 11-year experience in provision of prenatal diagnostic testing in Denmark observed miscarriage rates of 1.4% [95% confidence interval (CI), 1.3%-1.5%; n = 32,852] after amniocentesis compared with 1.9% (95% CI, 1.7%-2.0%; n = 31,355) after CVS.22 CVS has also been associated with increased risks for limb-reduction defects in historical cohorts. However, this seems to have low or absent risk when the procedure timing is after 9 weeks gestational age as there was no increase over the general population incidence of these anatomic defects.23 A comparison of amniocentesis and CVS is provided in Table 1.
Screening for Fetal Aneuploidy Using Biochemical Markers Given the described risks of prenatal diagnostic testing, many patients will opt for screening methods over amniocentesis or CVS. Screening methodologies, before the introduction of NIPT, have relied on indirect assessments of risk (screening) for the most common autosomal trisomies of Down syndrome (trisomy 21), Edward syndrome (trisomy 18), and Patau syndrome (trisomy 13). These screening algorithms utilized associated maternal blood biomarkers to provide a numerical risk assessment of these fetal conditions in each distinct pregnancy.
Noninvasive Prenatal Detection of Aneuploidy TABLE 1.
215
Comparison of Prenatal Diagnostic Tests
Comparison
Timing of procedure Fetal loss rate Method of karotype Approach Complications
Cytogenetic result accuracy
Chorionic Villus Sampling (CVS)
Amniocentesis
10-13 wk 1.9% (95% CI, 1.7%-2.0%) Direct and culture from villi Transcervical or transabdominal Miscarriage Postprocedure bleeding (7%-10%) Infection (0.3%) PROM/anhydramnios Maternal cell contamination Mosaicism Rh isoimmunization Limb-reduction defects (1%-2% if performed at
Noninvasive Prenatal Detection of Aneuploidy CHRISTOPHER ROBINSON, MD,* DIRK VAN DEN BOOM, PhD,w and ALLAN T. BOMBARD, MDw z *Department of Obstetrics and Gynecology/Division of MaternalFetal Medicine, University of Virginia, Charlottesville, Virginia; w Sequenom Inc.; and z Department of Obstetrics and Gynecology/ Division of Reproductive Medicine, University of California San Diego, San Diego, California Abstract: Noninvasive prenatal testing (NIPT) uses cell-free fetal DNA from the plasma of pregnant women to provide valuable information about the potential risks for fetal aneuploidy. This article provides a historical overview of both invasive diagnostic testing and serum screening approaches, both biochemical and the newer molecular noninvasive prenatal testing assays, used to identify patients who would be best served by invasive testing. Key words: noninvasive prenatal testing, NIPT
which remains the gold-standard for prenatal aneuploidy diagnosis at the time of this article. Understanding the historical advances in prenatal aneuploidy detection alongside advances in biomolecular science and bioinformatics in the past decade, can provide the context for a full understanding of the role and potential for use of new, molecular approaches to noninvasive methodologies in the prenatal detection of aneuploidy, known currently as noninvasive prenatal testing (NIPT)—and the subject of this chapter.
Prenatal aneuploidy detection and screening has been an area where major advances in technology have led to improved detection and accuracy over the past few decades. Recently, advances in genomic sequencing and bioinformatics have led to aneuploidy detection techniques that are approaching the detection rate and accuracy seen in amniocentesis and chorionic villus sampling-derived karyotype analysis,
Detection of Fetal Aneuploidy: A Brief Overview of Screening and Diagnosis Historically, prenatal diagnosis began to expand dramatically with growing interest in pregnancies carrying a fetus with Down syndrome. Down syndrome is a genetic disorder caused by the presence of all or part of an extra 21st chromosome
Correspondence: Allan Bombard, MD, Sequenom Inc., San Diego, CA. E-mail: [email protected] D.V.D.B. and A.T.B. are full-time employees of Sequenom, Inc. The other author has no conflict of interest to declare. CLINICAL OBSTETRICS AND GYNECOLOGY
210 | www.clinicalobgyn.com
/
VOLUME 57
/
NUMBER 1
/
MARCH 2014
Noninvasive Prenatal Detection of Aneuploidy (trisomy 21 or T21). It is named after John Langdon Down, the British physician who described the clinical features in 1866. The clinical disorder was identified as most commonly being due to an extra copy of chromosome 21 (trisomy) by Lejeune in 1959, and is characterized by a combination of major and minor physical features. Often, Down syndrome is associated with some impairment of cognitive ability and physical growth as well as hallmark physical features. Down syndrome can be identified during pregnancy or at birth. Trisomy 21 is the most common chromosomal aneuploidy in live born infants. The overall incidence is approximately 1 in 800 births in the general population but this risk increases to 1 in 35 term births for women aged 45 years. Advanced maternal age is only one factor contributing to the increased risk. When other factors, such as positive serum biochemical screening results or fetal ultrasound indicative of aneuploidy or previous or family history of aneuploidy are included, the incidence can be as high as 1 in 15 live born infants. The presence of any one of these risk factors places the pregnancy in the category of high risk for Down syndrome. Prenatal fetal assessment and testing can be provided through both invasive and noninvasive methodologies. Before the development of serum biochemical screening tests in the 1980s, aneuploidy was detected exclusively through invasive technology using genetic amniocentesis or chorionic villus sampling (CVS) to provide a sample of the fetal genotype for analysis.1 Invasive testing, which began almost 5 decades ago consists of CVS, which can be performed between 9.5 and 12.5 weeks’ gestation, or genetic amniocentesis that can be performed most commonly after 14 weeks’ gestational age.2 Analyses on specimens obtained by these invasive tests, owing to superior sensitivity and low false-positive rates, are considered
211
diagnostic and therefore the gold-standard in prenatal diagnosis. These surgical procedures preceded the availability of noninvasive assessment of fetal risk for certain heritable disorders. However, due to a strong desire on the part of patients and their physicians, less risky, noninvasive approaches evolved. Noninvasive screening approaches involve the use of risk factors for fetal aneuploidy. The first such screening tests were advanced maternal age and prior history—also the first indications for invasive diagnostic testing. Subsequently, with the recognition that abnormalities in the levels of certain biochemical markers could provide better identification of those women at an increased risk, the introduction of serum markers was soon followed by advances in fetal ultrasound that could also substantially aid in the identification of pregnancies at risk.1 The identification and quantification of specific, indirect fetal biomarkers found in the maternal serum presaged the advent of novel, new target assays in the maternal plasma that now constitute the latest advances in NIPT. When present in concentrations outside of the normal ranges for varying gestational ages, pregnant women who manifest positive serum biochemical screening tests were identified as patients who might be at an increased risk for fetal disease and who would derive the greatest benefit from invasive diagnostic testing, despite the risks of such procedures. Although these serum biochemical screening tests offer 70% to 90% detection, they are compromised by a 2.5% to 5% screen (usually false)-positive rate. Before the advent of NIPT there existed 4 principle risk factors for fetal aneuploidy: personal or family history; advanced maternal age; target serum analytes, biomarkers associated with aneuploidy; and ultrasound findings indicative of structural anomalies. A patient who has been identified as being at an www.clinicalobgyn.com
212
Robinson et al
increased risk for fetal aneuploidy because of the presence of one or more of these risks was traditionally felt to be a candidate for invasive prenatal diagnosis. This changed with the ACOG Committee Opinion #77, issued in January 2007 and which, for the first time, suggested that given adequate informed consent, any patient could be offered invasive diagnostic testing.3 Since then, the standard-ofcare has been that after counseling and the granting of informed consent, patients found to be at an increased risk may opt a priori for invasive diagnostic testing that provides fetal tissue diagnosis with a very high sensitivity and low false-positive rates. This approach is far more definitive than the alternative noninvasive prenatal serum biochemical screening methodologies that have a lower detection and higher false-positive rates for aneuploidy. Unlike invasive procedures, which carry a small but definite risk for fetal and maternal complications, noninvasive serum biochemical testing approaches have no such risks—but have poorer predictive values. Patients therefore are presently urged to consider the risks and benefits of both such approaches—noninvasive serum biochemical screening tests, with relatively poor sensitivity and specificity but little risk of fetal loss, and invasive diagnostic testing, with excellent sensitivity and specificity but the small but real risk for both fetal loss and maternal complications. The value proposition presented by NIPT is the ability to dramatically improve the sensitivity and specificity of risk assessment by noninvasive approaches to a level afforded by invasive testing, whereas retaining the safety of simple phlebotomy and avoiding risks to mother and baby in the vast majority of the patients.
Invasive Prenatal Diagnosis Invasive diagnostic sampling includes CVS in the late first trimester, genetic amniocentesis in the early second www.clinicalobgyn.com
trimester, or, less commonly, percutaneous umbilical blood sampling (PUBS) in the mid second trimester. CVS, genetic amniocentesis, and PUBS are invasive procedures, in that each involves inserting instruments into the uterus, and therefore add a small risk to the pregnancy of causing fetal injury or miscarriage. All 3 sampling methods are followed by karyotyping with or without fluorescence in situ hybridization (FISH), microarray and/or quantitative fluorescent polymerase chain reaction (QF-PCR), which includes amplification, detection, and analysis of chromosome-specific DNA sequences. The analyses from specimens obtained by these invasive diagnostic procedures are presently the only way to confirm a diagnosis of Down syndrome, or other abnormalities, and are usually offered to families who either screen positive by serum biochemical means or may have an increased chance of having a child with a chromosome or other heritable abnormality that is amenable to laboratory diagnosis. Noninvasive prenatal diagnostic processes have, as a result, been a longstanding research theme in prenatal medicine. In January 2007, the American Congress of Obstetricians and Gynecologists issued an updated Practice Bulletin that recommends that all pregnant women be offered both screening and the option of invasive diagnostic testing for fetal trisomy 21.4 It is well appreciated that the prevalence of fetal chromosomal aneuploidy varies as a function of both maternal and gestational age.5 For example, the prevalence of fetal chromosomal abnormalities exceeds 50% in first trimester, noted in spontaneous abortions.6 By contrast, at term, fetal aneuploidy can be found in 6% to 13% of stillbirths.7 Chromosomal abnormalities that are compatible with extrauterine life, but with significant morbidity and mortality, occur in B0.6% of newborns.8 Thus, invasive procedures—CVS, genetic amniocentesis, and PUBS—are surgical
Noninvasive Prenatal Detection of Aneuploidy procedures used to obtain fetal or fetally derived tissues for the detection of chromosomal abnormalities and have been key in enabling the assessment of the prenatal, intrauterine fetal condition and state. Although CVS and amniocentesis are common first and second trimester, respectively, surgical procedures, cordocentesis, provides a primary advantage of rapid karyotype analysis within 24 to 48 hours after procedure. However, it is rarely utilized owing to higher pregnancy loss rates. Alternative methods for rapid detection of common aneuploidies by FISH and QF-PCR are now routinely used in the United States and Europe, facilitating a more rapid result for patients undergoing CVS and amniocentesis. As mentioned above, the risk for fetal loss associated with these invasive procedures, driving the search for noninvasive alternatives with better performance than serum biochemical screening tests. Amniocentesis was the first invasive procedure developed to evaluate the fetal condition—whether metabolic or genetic. Amniocentesis has been reported as early as 1877 when Prochownick, Von Schatz, and Lambi performed the procedure in the third trimester of pregnancy. A single report from 1919 described the removal of amniotic fluid due to hydramnios in a third trimester pregnancy.9 Bevis10 in 1953 used amniotic fluid obtained by amniocentesis every 2 weeks to assist in the management and prediction of severity of Rh alloimmunization. The initial use of amniocentesis in the detection of genetic disease was described in 1956 in the journal Nature.2 Fuchs and Riis reported the ability to detect fetal sex based on the presence or the absence of the Barr body among cells obtained at amniocentesis. The ability to determine fetal sex by amniocentesis would lead to the use of invasive testing for the antenatal detection of sex-linked hereditary disorders such as Hemophilia A (1960) and Duchenne muscular dystrophy (1964).11
213
Nadler and Gerbie went on to publish the landmark article entitled ‘‘Role of amniocentesis in the intrauterine detection of genetic disorders’’ in the New England Journal of Medicine in 1970 where they described 162 amniocentesis procedures with a 97% success rate for chromosome analysis with a single amniocentesis procedure.12 This documented experience would form the basis for the utilization of invasive prenatal detection of the fetal genetic state in the second trimester. Around the same time, CVS was shown to be an alternative mechanism for assessment of the fetal state.13,14 The initial attempts at CVS were performed in 1968 with a rigid 5-mm endoscope with direct visualization of the chorion for direct biopsy and retrieval of villi. This initial approach in 1968 by Mohr in Scandinavia was successful in over 96% of the attempts but was complicated by unacceptable rates of bleeding, infection, and failed cultures.15 As a result, this methodology for CVS was abandoned as amniocentesis was less complicated and had a higher success rate for completed karyotype analysis. Ward et al16 would use ultrasound guidance with the passage of a transcervical catheter in 1983 to successfully biopsy the chorionic plate with 89% success. This methodology would be further refined into the modern CVS procedure for prenatal diagnosis. When considering prenatal diagnosis, there are many potential indications for performing amniocentesis or CVS in providing information concerning the fetal intrauterine state. Patients may desire testing for aneuploidy, single-gene defects, neural tube defects, or for confirmation of fetal blood type in cases of possible alloimmunization risk. When considering the totality of cases undergoing prenatal diagnostic procedures with amniocentesis or CVS, the majority of the procedures (>90%) are performed for aneuploidy detection among women who desire this information or who www.clinicalobgyn.com
214
Robinson et al
are deemed at high risk for aneuploidy. The ACOG supports prenatal diagnostic intervention availability for all women who desire this testing regardless of perceived/ actual risk or maternal age.4 An important aspect of prenatal diagnostic testing involves the pretest counseling regarding the risks and benefits of amniocentesis and CVS. CVS has the advantages of offering first trimester prenatal diagnostic karyotype information and can be performed directly and provide rapid results in 2 to 4 hours after procedure with direct preparation or after tissue culture for 48 to 72 hours with results within a week. There is a 99.7% rate of accuracy of cytogenetic results with CVS obtained chorionic villus material among more than 6000 patients enrolled in the United States Collaborative Study. This group found that a repeat procedure was necessary in 1.1% of patients due to maternal cell contamination or mosaicism noted in the culture.17 As CVS involves biopsy of the chorionic plate, contamination of the biopsy material can include maternal decidual tissue that can lead to erroneous results. This can be minimized through ensuring adequate villus tissue in the specimen and maternal cell contamination analysis to examine for polymorphisms that would allow distinction between maternal and fetal cells. Mosaicism also complicates around 1% of CVS specimens.17,18 When this is found, consideration of whether this is due to culture, confined placental mosaicism, or representative of the actual fetal state must be considered. To confirm this, a repeat procedure is often needed that is most commonly an amniocentesis. Amniocentesis has the benefit of avoiding the higher potential for maternal cell contamination and culture-based mosaicism seen in CVS, but carries the disadvantage of being performed in the second trimester (generally 15 to 20 wk’) that would not allow for early pregnancy termination if desired with an abnormal result. www.clinicalobgyn.com
CVS and amniocentesis have been examined with regard to fetal loss rates associated with each procedure. The overall rate of prenatal loss with a CVS procedure is greater than that of amniocentesis, though, this is most likely due to the increased background pregnancy loss rates seen at 6 to 16 weeks gestational age.19 Procedure-related loss rates for amniocentesis have been reported to be between 1/300 and 1/500 in the literature with even lower risks in more contemporary reports.20,21 A large, national registry describing an 11-year experience in provision of prenatal diagnostic testing in Denmark observed miscarriage rates of 1.4% [95% confidence interval (CI), 1.3%-1.5%; n = 32,852] after amniocentesis compared with 1.9% (95% CI, 1.7%-2.0%; n = 31,355) after CVS.22 CVS has also been associated with increased risks for limb-reduction defects in historical cohorts. However, this seems to have low or absent risk when the procedure timing is after 9 weeks gestational age as there was no increase over the general population incidence of these anatomic defects.23 A comparison of amniocentesis and CVS is provided in Table 1.
Screening for Fetal Aneuploidy Using Biochemical Markers Given the described risks of prenatal diagnostic testing, many patients will opt for screening methods over amniocentesis or CVS. Screening methodologies, before the introduction of NIPT, have relied on indirect assessments of risk (screening) for the most common autosomal trisomies of Down syndrome (trisomy 21), Edward syndrome (trisomy 18), and Patau syndrome (trisomy 13). These screening algorithms utilized associated maternal blood biomarkers to provide a numerical risk assessment of these fetal conditions in each distinct pregnancy.
Noninvasive Prenatal Detection of Aneuploidy TABLE 1.
215
Comparison of Prenatal Diagnostic Tests
Comparison
Timing of procedure Fetal loss rate Method of karotype Approach Complications
Cytogenetic result accuracy
Chorionic Villus Sampling (CVS)
Amniocentesis
10-13 wk 1.9% (95% CI, 1.7%-2.0%) Direct and culture from villi Transcervical or transabdominal Miscarriage Postprocedure bleeding (7%-10%) Infection (0.3%) PROM/anhydramnios Maternal cell contamination Mosaicism Rh isoimmunization Limb-reduction defects (1%-2% if performed at
