This document addresses cell-free fetal DNA-based prenatal testing for fetal aneuploidies (including fetal sex chromosome aneuploidies), fetal sex determination and microdeletions.
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Cell-free fetal DNA-based prenatal screening for fetal aneuploidy (trisomy 13, 18, and 21) is considered medically necessary for women with a current single gestation pregnancy and genetic counseling, which encompasses all of the following components, has been performed:
Cell-free fetal DNA-based prenatal testing for fetal sex determination is considered medically necessary for singleton pregnancies at increased risk of a sex (X)-linked condition or congenital adrenal hyperplasia and genetic counseling, which encompasses all of the following components, has been performed:
Not Medically Necessary:
Cell-free fetal DNA-based prenatal testing for fetal sex determination is considered not medically necessary for pregnancies without an increased risk of a sex (X)-linked condition or congenital adrenal hyperplasia.
Investigational and Not Medically Necessary:
Cell-free fetal DNA-based prenatal screening for fetal aneuploidy (trisomy 13, 18, 21) is considered investigational and not medically necessary for individuals not meeting the criteria above, including women with a current multiple gestation pregnancy.
Cell-free fetal DNA-based prenatal testing for fetal sex chromosome aneuploidies is considered investigational and not medically necessary.
Cell-free fetal DNA-based prenatal testing for microdeletion syndromes is considered investigational and not medically necessary.
Cell-free fetal DNA-based prenatal testing is considered investigational and not medically necessary for all other indications.
New developments in screening methods for fetal chromosomal abnormalities have increased the number of options available to pregnant women. Historically, screening options in the first trimester included nuchal translucency testing, along with measurement of pregnancy-associated plasma protein A and human chorionic gonadotropin. Screening options in the second trimester included serum screening using triple or quadruple bio-marker screening and ultrasonography. Individuals also had the option of choosing a combination of first- and second-trimester screening in an integrated, stepwise sequential or contingent sequential fashion. For women who did not present until the second trimester, the quadruple screen was recommended.
Aneuploidies of chromosomes 13, 18, 21, X, and Y are the most frequently represented chromosome anomalies detected in prenatal specimens. Historically, the standard of care for the prenatal diagnosis of chromosome anomalies is amniocentesis, chorionic villus sampling (CVS) or cordocentesis. These invasive tests carry a significant risk of pregnancy loss and are generally offered only to those women at high risk of carrying an affected fetus. Because of the high risk of pregnancy loss, researchers are exploring the use of non-invasive methods to prenatally diagnose fetal chromosomal anomalies. While these newer techniques may help avoid invasive genetic testing, it is important to note that genetic testing should be offered in a setting with adequately trained health care professionals to provide appropriate pre-test and post-test counseling. An individual with a positive cell-free fetal DNA test result should be offered invasive prenatal diagnostic testing (chorionic villus sampling or amniocentesis) for confirmation of test results.
Approximately 10% of circulating cell-free DNA in maternal plasma at the end of the first trimester is of fetal origin (Handley 2010). Researchers are investigating the use of fetal-specific DNA sequences in maternal plasma as a non-invasive means to detect fetal aneuploidies such as trisomy 13, 18 and 21. These tests involve "massively parallel DNA sequencing" (also known as MPS) or "next generation DNA sequencing" to identify an increased or decreased representation of chromosomes associated with fetal aneuploidies. During these testing methods, millions of DNA fragments (fetal and maternal) are sequenced and the number of sequences specific to each chromosome is determined. The proportion of sequence fragments representing these chromosomes has been shown to be greater in the maternal blood of fetuses with trisomy. The test involves the extraction and purification of cell-free DNA from maternal plasma and sequence analysis of DNA fragments. For each fragment, a short sequence (35-50 base pairs) or "tag" is identified and compared with a human genome reference standard to identify its chromosome of origin. The analysis creates a "z-score" representing the number of standard deviations from the mean (Chiu, 2009).
One of the shortcomings of MPS is that it is not selective in the chromosomal origin of the sequenced DNA fragments. As an example, chromosome 21 represents only approximately 1.5% of the human genome; therefore, when using MPS, it is necessary to sequence many millions of fragments to ensure a sufficient count of chromosome 21. As an alternative to overcome these limitations, researchers are investigating another technique, digital analysis of selected regions (DANSR™ ), which entails selective sequencing of loci from only the chromosomes that are under investigation. Researchers have also investigated the use of the fetal-fraction optimized risk of trisomy evaluation (FORTE) by extending the process of chromosome-selective sequencing to assay nonpolymorphic and polymorphic loci, where fetal alleles differ from maternal alleles. This allows the simultaneous determination of chromosome proportion and fetal fraction (Ashoor, 2012; Sparks, 2012a; Sparks, 2012b).
Chiu and colleagues (2008) used these techniques to non-invasively detect fetal trisomy 21. The study tested 28 first and second trimester maternal plasma samples, 14 known to be trisomy 21 and 14 known to be euploid fetuses using full karyotyping. All 14 trisomy 21 and 14 euploid fetuses were correctly identified suggesting that this type of sequence analysis of cell-free DNA in maternal plasma might be used to detect trisomy 21 during the first trimester.
Ehrich and colleagues (2011) evaluated a multiplexed (multiple simultaneous sample analysis) MPS assay for non-invasive trisomy 21 detection using circulating cell-free fetal DNA. A total of 480 archived plasma samples from high-risk pregnant women who had undergone invasive prenatal testing were analyzed. Of the 480 prospectively collected samples, 449 samples could be evaluated. Investigators were blinded to karyotype status of the samples. All 39 (100%) of trisomy 21 samples were identified using sequence analysis of maternal plasma DNA. One (0.2%) of the 410 euploid samples was misidentified as trisomy 21. The authors reported a sensitivity of 100% (95% confidence interval [CI], 89%-100%) and a specificity of 99.7% (95% CI, 98.5% to 99.9%). The researchers concluded that this study demonstrated that plasma DNA sequencing is a viable method for the non-invasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.
Using a modified testing protocol and different sequencing platform, Chiu and colleagues (2011) analyzed 86 samples of women at high risk for trisomy 21 based on current prenatal screening testing and who subsequently underwent invasive prenatal testing and were found to carry fetuses with trisomy 21. Using a z-score cutoff of 3 and a 2-plex (2 specimens analyzed simultaneously) Genome AnalyzerIIx DNA sequencing platform, sensitivity was 100%, specificity 97.9% with a positive predictive value (PPV) of 96.6% and negative predictive value (NPV) of 100%. Diagnostic performance was much lower using an 8-plex testing protocol with a sensitivity of only 79.1%, specificity 98.9%, PPV 91.9%, and NPV 96.9%. The authors compared their results with the reported performance of the standard three marker screening test with an NPV of 95% (5% false positive rate). They suggest that the data from this study indicate the main value of maternal plasma DNA sequencing is to rule out trisomy 21 and could be used to stratify pregnancies whose risk for trisomy 21 warrants invasive amniocentesis or chorionic villus sampling (CVS).
Palomaki and colleagues (2011) reported the findings of a validation study of 4664 women at high risk for trisomy 21 (maternal age, bio-marker screening tests, and/or fetal ultrasound.) Fetal karyotyping was compared with DNA sequence analysis in 212 cases of trisomy 21 and 1484 matched euploid cases. Maternal blood samples were drawn and sent to two independent, CLIA-certified laboratories for testing. DNA was sequenced using a 4-plex protocol and the HiSeq 2000™ sequencing platform. Of the 4664 high-risk cases, 279 were excluded from analysis (inadequate specimen, multiple gestations, fetal death, and karyotype not available). Five percent (218) were found to have trisomy 21 based on karyotyping and 3930 were euploid. The analysis was limited to the first 212 trisomy 21 cases. Using a z-score of 3, trisomy 21 was detected in 209 of 212 cases. Sensitivity was reported as 98.6%, with a false positive rate of 0.2%. A subset analysis performed by an independent laboratory confirmed these results.
Researchers are also investigating if maternal cell-free fetal DNA sequencing has the capability to detect other aneuploidies, such as trisomy 18 and 13. Sehnert and colleagues (2011) investigated the ability of MPS of cell-free fetal DNA from maternal blood to detect trisomy 18. Blood samples were collected from 1014 female participants at 13 clinics in the United States before they underwent an invasive prenatal procedure. All samples were processed to plasma, and the DNA retrieved from 119 samples underwent MPS. Fifty-three sequenced samples came from women with an abnormal fetal karyotype. In order to minimize the intra- and inter-run sequencing variation, the researchers developed an algorithm by using normalized chromosome values from the sequencing data on a training set of 71 samples with 26 abnormal karyotypes. The classification process was then evaluated on an independent test set of 48 samples with 27 abnormal karyotypes. Mapped sites for chromosomes of interest in the sequencing data from the training set were normalized individually by calculating the ratio of the number of sites on the specified chromosome to the number of sites observed on an optimized normalizing chromosome (or chromosome set). Threshold values for trisomy or sex chromosome classification were then established for all chromosomes of interest, and a classification schema was defined. Sequencing of the independent test set led to 100% of the trisomy 21 (13 of 13) samples and trisomy 18 (8 of 8) samples being correctly identified. The algorithm also correctly identified the presence of trisomy 21 in 2 sets of twin pregnancies with at least 1 affected fetus and 1 case of trisomy 9. The authors concluded that MPS is capable of detecting multiple fetal chromosomal abnormalities from maternal plasma when an optimized algorithm is used.
In a study by Palomaki and colleagues (2012) and sponsored by the Women and Infants Hospital of Rhode Island in collaboration with Sequenom Center for Molecular Medicine, the performance of maternal cell-free fetal DNA sequencing for the detection of trisomy 18 and 13 was a secondary endpoint. Of the 1988 pregnancies with matched euploid controls, 212 samples were identified with trisomy 21, 62 samples with trisomy 18, and 12 samples with trisomy 13. Three trisomy 18 samples failed due to the fetal fraction being lower than the prespecified lower limit (4%). The detection rate for trisomy 18 among the interpreted samples was 59/59 (100%). Among the 12 pregnancies with trisomy 13, the detection rate for trisomy 13 was 11/12 (91.7%). One trisomy 13 sample was signed out as normal (false negative). Overall, testing failed to provide a clinical interpretation in 17 participants (0.9%); 3 of which had a trisomy 18 pregnancy.
Bianchi and colleagues (2012) prospectively examined the diagnostic accuracy of MPS (the verifi® Prenatal Test; Illumina, Inc., San Diego, CA) to detect whole chromosome fetal aneuploidy in maternal plasma. Blood samples were collected in a prospective, blinded study from 2882 female participants undergoing prenatal diagnostic procedures at 60 locations in the United States. An independent biostatistician selected all singleton pregnancies with any abnormal karyotype and a balanced number of randomly selected pregnancies with euploid karyotypes. From a pool of 2625 specimens, 532 (20%) maternal plasma specimens were tested. The verifi test correctly identified 89 of 89 cases (100%) of trisomy 21, 35 of 36 cases (97.2%) of cases of trisomy 18, 11 of 14 cases (78.6%) of trisomy 13 and 15 of 16 cases (93.8%) of monosomy X. The gender of fetuses was also determined with 232 of 233 cases (99.6%) of female fetuses being accurately identified and male fetuses correctly identified in 184 of 184 cases (100%). No false positives for aneuploidies of chromosomes 13, 18 or 21 were identified, but there was one specimen that was incorrectly identified as monosomy X. The researchers concluded that this test is highly accurate and can be incorporated into prenatal aneuploidy screening algorithms to reduce the incidence of invasive procedures.
Sparks and colleagues (2012b) explored the development of a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for the evaluation of fetal trisomy 21 and trisomy 18. A total of 298 pregnancies, including 39 trisomy 21 and 7 trisomy 18 confirmed fetal aneuploidies, were analyzed using DANSR. Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. The products from 96 separate subjects were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from low-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. At the lowest sequencing depth of 204,000 sequencing counts per sample, low-risk cases where distinguished from trisomy 21 and trisomy 18 cases. Increasing the sequencing depth to 410,000 counts per sample substantially improved separation of aneuploid and low-risk cases. A further increase of the sequencing depth to 620,000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by MPS approaches. The researchers concluded that DANSR enables highly accurate, cost efficient and scalable non-invasive fetal aneuploidy assessment.
Ashoor and colleagues (2012) assessed the prenatal detection rate of trisomy 21 and 18 and the false positive rate by chromosome-selective sequencing of maternal plasma cell-free DNA. In this nested case-control study, cell-free DNA was examined in plasma that was obtained at 11-13 weeks before chorionic villous sampling from 300 euploid pregnancies, 50 pregnancies with trisomy 21, and 50 pregnancies with trisomy 18. The laboratory personnel were blinded to fetal karyotype. Risk scores for trisomy 21 and 18 were assigned for 397 of the 400 samples that were analyzed. In all 50 cases of trisomy 21, the risk score for trisomy 21 in 47 cases was greater than or equal to 99%, and the risk score for trisomy 18 was less than or equal to 0.01%. In the 50 cases of trisomy 18, the risk score for trisomy 21 was less than or equal to 0.1%; for trisomy 18, the risk score was greater than or equal to 99% in 47 cases. In 3 of the 300 euploid pregnancies (1%), no risk score was provided because amplification and sequencing failed. In the remaining 297 cases, the risk score for trisomy 21 was less than or equal to 0.01%; for trisomy 18, the risk score was less than or equal to 0.1% in 295 cases, was 0.04% in 1 case, and was 0.23% in 1 case. Overall, the sensitivity was 100% (50/50 cases) for the detection of trisomy 21 and was 98% (49/50 cases) for the detection of trisomy 18; specificity was 100% (297/297 cases). The authors concluded that the DANSR assay with FORTE algorithm is a promising method for detecting fetal trisomy 21 and 18 from cell-free DNA in maternal blood during the first trimester of pregnancy. The authors also acknowledged that additional research is needed to investigate the accuracy of the test in intermediate-risk and low-risk pregnancies and to expand the spectrum of aneuploidies that could be detected by analysis of maternal plasma cell-free DNA.
In another study, Sparks and colleagues (2012a) evaluated the use of the DANSR assay and FORTE algorithm for the prenatal evaluation of risk for fetal trisomy 21 and trisomy 18 using cell-free DNA obtained from maternal blood. The researchers assayed cell-free DNA from a training set and a blinded validation set of pregnant females, consisting of 250 euploidy, 72 trisomy 21, and 16 trisomy 18 pregnancies. The researchers then used the DANSR assay in combination with FORTE algorithm to determine trisomy risk for each participant. Overall, 163/171 subjects in the training set passed quality control criteria. Using a Z statistic, 35/35 trisomy 21 cases and 7/7 trisomy 18 cases had Z score greater than 3 and 120/121 euploid cases had Z score less than 3. FORTE produced an individualized trisomy risk score for each participant and correctly identified all trisomy 21 and trisomy 18 cases from euploid cases. All 167 subjects in the blinded validation set passed quality control and FORTE performance matched that observed in the training set correctly identified 36/36 trisomy 21 cases and 8/8 trisomy18 cases from 123/123 euploic cases. The authors concluded that digital analysis of selected regions in conjunction with FORTE enable accurate, scalable non-invasive fetal aneuploidy detection.
Norton (2012) reported on the findings of a multicenter cohort study involving 3228 pregnant women with a gestational age at or equal to 10 weeks with singleton pregnancies who were evaluated for the presence of trisomy 21 or trisomy 18. Subjects were classified as either high- or low-risk of aneuploidy based on the results of DANSR and FORTE analysis of cell-free fetal DNA from maternal blood samples. A total of 81 subjects were classified as high-risk for trisomy 21. There was one false positive result among the 2888 normal cases, for a sensitivity of 100% and a false positive rate of 0.03%. Thirty-eight subjects were identified as having trisomy 18 present, and 37 were classified as high-risk. There were two false positive results among the 2888 normal cases, for a sensitivity of 97.4% and a false positive rate of 0.07%. It was concluded that chromosome-selective sequencing of cell-free DNA and application of an individualized risk algorithm was effective in the detection of fetal trisomy 21 and trisomy 18.
Testing for trisomy 21 and trisomy 18 was conducted in 11,105 women with singleton pregnancies at a gestational age of 12 weeks (Dan, 2012). A total of 190 subjects were classified as positive, including 143 with trisomy 21 and 47 with trisomy 18. Based on confirmatory karyotype testing and fetal outcome data, the authors observed one false positive case of trisomy 21, one false positive case of trisomy 18, and no false negative cases, indicating 100% sensitivity and 99.96% specificity for the detection of both trisomy 21 and trisomy 18.
A study by Canick (2012) reported on the use of MPS to identify the presence of trisomy 21, trisomy 18, and trisomy 13 in 4664 pregnant women with multiple gestations and at high risk of aneuploidy. Seven twin pregnancies affected by Down syndrome were identified by their karyotypes; two in which both fetuses were affected and five in which just one fetus was affected. One twin pregnancy discordant for trisomy 13 was also identified. The authors reported a detection rate of 100% and a false positive rate of 0% for trisomy 21 and trisomy 13.
The American College of Medical Genetics and Genomics has indicated that noninvasive prenatal screening using cell-free DNA should not be used to screen for autosomal aneuploidies other than those involving chromosomes 13, 18, and 21 (Gregg, 2016).
Compared with high-risk pregnancies, there is limited evidence evaluating the screening accuracy of cell-free fetal DNA prenatal testing in low-risk pregnancies. Although some studies suggest the test performance of cell-free fetal DNA aneuploidy screening in a low-risk population is similar to results reported for high-risk pregnancies, these findings are weakened by study design limitations. Bianchi and colleagues (2014) conducted an industry-sponsored study to evaluate the performance of verifi, a cell-free DNA prenatal test, in a general population sample (n=2042; mean age, 29.6 years). Enrollees were women with singleton pregnancies at 8 or more weeks of gestation, who were undergoing standard aneuploidy screening (various serum biochemical assays, with or without nuchal translucency measure). Pregnant women in any trimester of pregnancy were eligible for inclusion. Study authors compared the false positive rate of cell-free DNA testing with standard prenatal screening methods as the primary endpoint. The principal reference standard was the newborn physical exam (97%). Only 3% of cases were confirmed with karyotype analysis. Screening was incomplete for 39 women and 10 women were eliminated due to inadequate blood sampling. There were 5 cases of T21 (Downs), which were all correctly identified using both cell-free fetal DNA and standard screening.
Results showed that for the detection of T21, cell-free fetal DNA testing resulted in a significantly lower false positive rate (0.3%) compared with conventional testing (3.6%) (p<0.001). For detection of trisomy 18, the false positive rate was also significantly lower compared with conventional testing (0.2% vs. 0.6%; p=0.03). However, the study was not sufficiently powered to compare detection rates between cell-free fetal DNA and standard methods. It is also notable that the study included pregnant women in all trimesters (39.7% first trimester, 21.9% second trimester, 28.5% third trimester). Because fetal DNA amounts are higher in later stages of pregnancy, the study results may not reflect test performance in the first trimester when testing would typically be offered. The study compared cell-free fetal DNA screening with various standardized test protocols. However, only 2.8% of women had "fully integrated screening" (first trimester serum markers plus nuchal translucency, and second trimester serum markers), which is the testing method believed to have the best performance and lowest false positive rate (Malone, 2005). This study detected 5 trisomy 21 cases out of 1909 low-risk women, a rate nearly twice the expected rate in a low-risk cohort.
In an industry sponsored, prospective, blinded study (NCT01511458), Norton and colleagues (2015) carried out a head-to-head comparison of standard first-trimester screening versus cell-free DNA testing in a population of average risk, single gestation women who received routine obstetric care at centers in the USA, Canada and Europe. The researchers assigned pregnant women presenting for aneuploidy screening at 10 to 14 weeks of gestation to undergo both standard screening (with measurement of nuchal translucency and biochemical analytes) and cell-free DNA testing. Participants received the results of standard screening, however; the results of cell-free DNA testing were blinded. Determination of the birth outcome was determined using diagnostic genetic testing or newborn examination. A total of 18,955 individuals were enrolled in the study and 15,841 were available for analysis. Cell-free fetal DNA testing identified all 38 cases (100% [95% CI, 90.7 to 100]) of trisomy 21 identified in the study, while standard screening identified only 30 of these cases (78.9% CI, 62.7 to 90.4; p=0.008). False positive rates were 0.06% (95% CI, 0.03 to 0.11) in the cell-free DNA group versus 5.4% (95% CI, 5.1 to 5.8) in the standard-screening group (p<0.001). The positive predictive value of screening with cell-free DNA was 80.9% (66.7 to 90.9) versus 3.4 (2.3 to 4.8) for standard screening (p<0.001). Overall, cell-free DNA testing for trisomy 21 outperformed standard screening using nuchal translucency measurement and biochemical analytes. False positives were also identified for trisomies 13 and 18 but the study was not adequately powered to examine the performance of cell-free DNA analysis to screen for these conditions. The authors also reported an unbalanced chromosome rearrangement or trisomy in 2.7% of the pregnancies that did not yield a cell-free DNA result.
Zhang and colleagues (2015) reported the clinical performance of massively parallel sequencing-based non-invasive prenatal testing (NIPT) in detecting trisomies 21, 18 and 13 in over 140,000 clinical samples and compared its performance in both, low-risk and high-risk pregnancies. Between January 2012 and August 2013, a total of 147,314 NIPT requests to screen for fetal trisomies 21, 18 and 13 using low-coverage whole-genome sequencing of plasma cell-free DNA were received. The results were validated using karyotyping or follow-up of clinical outcomes. Of the 146,958 samples tested, results were available in 112,669 (76.7%). Repeat blood sampling was necessary in 3213 cases and a total of 145 had test failure. Aneuploidy was confirmed in 720/781 of the cases positive for trisomy 21, 167/218 of the cases positive for trisomy 18 and 22/67 of the cases positive for trisomy 13 on NIPT. A total of 9 false negatives were identified, including 6 cases of trisomy 21 and 3 cases of trisomy 18. The sensitivity of NIPT was 99.17%, 98.24% and 100% for trisomies 21, 18 and 13, respectively. The specificity was 99.95%, 99.95% and 99.96% for trisomies 21, 18 and 13, respectively. The study did not identify any significant difference in test performance between the 72,382 high-risk participants and the 40,287 low-risk participants (sensitivity, 99.21% vs 98.97%; p=0.82); specificity was 99.95% vs 99.95% (p=0.98). The major factors contributing to false-positive and false-negative NIPT results were fetal/placental mosaicism and maternal copy number variant, but fetal fraction had no effect. A limitation of this study was the incomplete follow-up of NIPT results, which could possibly introduce bias into the performance evaluation. In the group of NIPT-positive cases, only 66.8% of the participants provided the result of a confirmatory diagnosis, mostly because they declined to provide clinical outcomes (17.9%) or they elected pregnancy termination (13.0%).
Several professional organizations have published documents addressing DNA-based prenatal testing for fetal aneuploidy. In 2013, the International Society for Prenatal Diagnosis (ISPD) issued a position statement regarding prenatal diagnosis of chromosomal abnormalities which indicated that while maternal cell-free DNA screening is an emerging technology that can provide highly effective prenatal screening for Down syndrome, trisomy 18, and possibly trisomy 13 in high-risk women, it should not be considered a replacement for the analysis of amniotic fluid cells or CVS. The position statement also pointed out that the information is insufficient to determine how well the test will perform in multiple gestation pregnancies that are discordant for trisomy and its efficacy in low-risk populations has not yet been fully demonstrated (Benn, 2013). However, this document was replaced by a more recent ISPD position statement which considers cell-free fetal DNA screening as a primary test for all pregnant women an appropriate testing protocol option (Benn, 2015).
Also in 2013, the National Society of Genetic Counselors (NSGC) issued a position statement that supports the use of non-invasive prenatal testing and non-invasive prenatal diagnosis (NIPT/NIPD) as an option for individuals whose pregnancies are considered to be at an increased risk for certain chromosome abnormalities. The NSGC also states that noninvasive prenatal test results should not be considered diagnostic at this time, "and any abnormal results should be confirmed through a conventional prenatal diagnostic procedure, such as chorionic villus sampling or amniocentesis." With regard to MPS, the NSGC states that while there are clinical studies demonstrating the ability of MPS to detect abnormalities in chromosomes 13, 18 and 21:
NIPT has not yet been proven efficacious in detecting other chromosomal abnormalities or single-gene disorders. NSGC recommends that pretest counseling for NIPT include information about the disorders that it may detect, its limitations in detecting these conditions, and its unproven role in detecting other conditions (Devers, 2013).
In 2014, the Society for Maternal and Fetal Medicine (SMFM) published a position statement regarding cell-free fetal DNA screening in low-risk women. The SMFM states that the currently available evidence regarding the use of cell-free fetal DNA for screening in a low-risk population is not sufficient enough to warrant a change to the current recommendations published by the American College of Obstetricians and Gynecologists (ACOG) and the SMFM. Additional clinical studies evaluating appropriate outcome measures are necessary before recommendations regarding non-invasive prenatal testing for aneuploidy in high-risk pregnancies can be expanded to include low-risk pregnancies.
In a joint opinion statement published in 2012, ACOG and the SMFM concluded that cell-free fetal DNA testing is one tool that can be used as a primary screening test in women at increased risk of aneuploidy. In 2015, ACOG replaced the 2012 document with a Committee Opinion on cell-free DNA screening for fetal aneuploidy which reviewed the advantages and limitations of the application of cell-free DNA screening in all pregnant women. ACOG concluded that after giving consideration to several factors including, but not limited to the performance of conventional screening methods and the limitations of cell-free DNA screening performance, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population (ACOG, 2015).
Consistent with the 2012 publication, in the 2015 document, ACOG re-affirmed that cell free fetal DNA testing is not recommended for women with multiple gestations because it has not been sufficiently evaluated in this group. While ACOG acknowledges that cell -free fetal DNA has tremendous potential as a screening tool, the specialty society also cautions that negative cell-free fetal DNA test results do not ensure an unaffected pregnancy. Although rare, false positive test results have been observed and reported in the literature. ACOG points out that "not only can there be false-positive test results, but a positive cell-free DNA test result for aneuploidy does not determine if the trisomy is due to a translocation, which affects the risk of recurrence." For this reason, individuals with a positive test result should be referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation of test results. ACOG also acknowledges that, in a small percentage of cases, a cell-free fetal DNA result will not be obtainable. Screening for microdeletions has not been validated in clinical studies so the true sensitivity and specificity of cell-free DNA screening for microdeletions is uncertain. Therefore, routine cell-free DNA screening for microdeletion syndromes is not recommended (ACOG, 2015).
In summary, several studies support the clinical validity of DNA-based testing of maternal plasma in women at high risk for a trisomy 21 pregnancy when compared with karyotype analysis, following amniocentesis or CVS (Ashoor, 2012; Chiu, 2008; Chiu, 2011; Ehrich, 2011; Fan, 2008; Palomaki, 2011). There are also studies supporting the clinical validity of DNA-based testing of maternal plasma in women at high risk for trisomy 13, 18 and trisomy X (Ashoor, 2012; Bianchi, 2012; Palomaki, 2012, Sparks, 2012a; Sparks, 2012b; Sehnert, 2011). Several of the studies used different protocols and non-standard sequencing platforms, software algorithms, and analyses. Although gestational age and maternal weight have been shown to affect test results, most studies did not control for these variables. Also, various test protocols were used in the studies, and several of the earlier studies were quite small and lacking in power. However, in spite of these limitations, ACOG and NSGC have concluded that there is sufficient evidence to support the use of cell-free fetal DNA-based non-invasive screening for aneuploidy in select individuals at high risk for fetal aneuploidy. These specialty organizations also recommend that any abnormal results be confirmed through conventional prenatal diagnostic procedure such as CVS or amniocentesis (ACOG, 2015; Benn, 2015; NSGC, 2012). The advantages of antenatal screening include increasing the odds of identifying an abnormal fetus and reducing the number of invasive diagnostic tests and procedure-related losses of normal fetuses. The disadvantage of screening is that not all aneuploid fetuses are identified with screening (Anderson, 2009). ACOG also cautions that cell-free DNA testing as a screening tool provides information regarding only trisomy 21, trisomy 18 and in some laboratories, trisomy 13. There is also the possibility that in a small percentage of cases, a DNA-based non-invasive prenatal screening result will not be obtainable (ACOG, 2012; ACOG, 2015; SMFM, 2014).
With regard to women at low-risk for aneuploidy, noninvasive cell-free DNA-based screening for fetal aneuploidy is considered as an acceptable screening option for fetal aneuploidy (trisomy 13, 18 and 21) in average-risk women carrying a single gestation. Individuals undergoing cell-free DNA-based testing should be advised of the limitations and benefits of this screening paradigm in the context of alternative screening and diagnostic options. Individuals with positive non-invasive cell-free DNA screening results should undergo confirmatory diagnostic prenatal testing (such as amniocentesis or chorionic villus sampling) (ACOG, 2015; ACOG, 2016; Gregg, 2016).
Fetal Sex Determination
Prenatal fetal sex determination is generally performed for women who are at risk of having a child with a serious genetic disorder affecting a particular sex. This includes women who are carriers of X-linked genetic disorders such as Duchenne muscular dystrophy (DMD) and adrenoleukodystrohy (ALD) and where fetal sexing is employed to guide decisions about invasive testing. Prenatal fetal sex determination may also be performed for carriers of hemophilia, where it can inform management of labor and delivery of confirmed and 'at risk' male pregnancies. In addition, fetal sex determination is used for conditions associated with ambiguous development of the external genitalia, such as congenital adrenal hyperplasia (CAH), where treatment with maternal steroids early in pregnancy can reduce the level of virilisation in female fetuses. Chorionic villus sampling from 11 weeks or amniocentesis from 15 weeks are invasive options which allow definitive genetic diagnosis, but both techniques carry a 1% risk of miscarriage. Invasive cytogenetic determination is considered the gold standard for ambiguous genitalia, X-linked conditions and singe-gene disorders such as CAH (Devaney, 2011; Heland, 2016; Hill, 2012; Lewis, 2012).
Traditionally, ultrasound has been method used for fetal sex determination. Several authors have explored the test performance of fetal ultrasound for sex determination with varying results. Fetal sex determination can be performed by ultrasound as early as 11 weeks gestation, although not reliably. Based on the findings of a review by Odeh and colleagues (2009), fetal sex cannot be determined by ultrasound examination in 7.5% to 50% of pregnancies at 11 weeks' gestation. However, this number decreased to 3% to 24% at 13 weeks. When reported, the sex determination is incorrect as much as 40% of the time at 11weeks, but by 13 weeks, accuracy (when reported) is close to 100%. In a prospective study by Chelli and colleagues (2009), the authors discovered that fetal sex could be determined by ultrasound 90% of the time, with 86% accuracy from 11 to 14 weeks gestation. More recently, researchers have explored the use of NIPD using cell-free DNA to determine the gender of the fetus. In the systematic review and meta-analysis conducted by Devaney and colleagues (2011), the researchers investigated the overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. A total of 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. The authors reported that sensitivity and specificity for detection of Y chromosome sequences was highest using RTQ-PCR after 20 weeks gestation. The authors also reported that urine tests as well as tests performed prior to 7 weeks gestation were unreliable. A limitation of this study and potential sources of bias is the use of a single database, which may have resulted in the omission of studies indexed elsewhere. Another limitation of the study was the relatively small studies included in the meta-analysis (Devaney, 2011).
NIPD using cell-free DNA is also being marketed to curious parents-to-be as a means of determining fetal sex for non-medical purposes. Currently, ultrasound examination is the standard non-invasive method of determining fetal sex. Use of NIPD using cell-free DNA for the purposes of sex determination for non-medical reasons is considered not medically necessary as information regarding the sex of the fetus which does not have any clinical impact of the health outcomes of the parent or fetus can be obtained via routine prenatal ultrasound when the intent is to identify fetal anomalies. ACOG acknowledges that some individuals may request cell-free DNA screening in order to obtain fetal sex information earlier than is possible with other methods such as ultrasound evaluation. ACOG recommends that individuals should be advised that cell-free DNA screening also assesses the risk of other trisomies and if that information is not desired, the screening should not be performed (ACOG, 2015).
It is also worth noting, that although NIPD plays an important role in the non-invasive prenatal diagnosis of selected conditions, cell-free fetal DNA-based testing does not eliminate the need for ultrasound studies (Gregg, 2016). The Blue Cross Blue Shield Technology Assessment provides a discussion of this issue and states:
The first trimester ultrasound scan is required to confirm gestational age and to determine whether the pregnancy is multiple, findings that provide necessary information for sequencing-based testing of cellfree fetal DNA. Ultrasound examination that details fetal anatomy in the second trimester is important for fetal risk assessment, and may provide indications of chromosomal abnormalities not currently detected by cell-free fetal DNA sequencing-based tests. Cell-free fetal DNA-based testing is also not a replacement for second trimester maternal screening for risk of neural tube defects (BCBSA, 2014).
In summary, NIPD for fetal sex determination provides an important alternative to cytogenetic determination (amniocentesis, chorionic villus sampling) and can improve the safety of medical care by reducing the need for invasive fetal diagnostic tests. The overall performance of noninvasive fetal sex determination using maternal blood can be high, provided that the blood sample is taken at a time during pregnancy when sufficient cell-free fetal DNA is present (7 weeks gestation or later). NIPD can feasibly be performed from as early as 7 weeks gestation, has been shown to be more than 99% accurate and, because it is non-invasive, does not carry the risk of miscarriage. Furthermore, NIPD has been estimated to reduce the need for invasive procedures by up to 50%. NIPD using cell-free DNA for sex determination can be useful in the clinical setting for early detection of fetuses at risk for sex-linked disorders which require follow-up testing. When used for fetal sex determination, NIPD offers several advantages over ultrasound and invasive testing. NIPD using cell-free DNA is a reliable non-invasive means to determine fetal sex without incurring the risk of unintended fetal losses that is associated with invasive procedures. NIPD for fetal sex determination is not appropriate when it is performed solely for non-clinical purposes (such as when a fetus with a sex linked genetic defect is not suspected or to provide the parents with the convenience of knowing the gender of the fetus sooner than can be determined by a routine prenatal ultrasound) (ACOG, 2015; Allyse, 2015; Devaney, 2011; Lewis, 2012).
Fetal Sex Chromosome Aneuploidy (SCA) Screening
Sex chromosome disorders are part of a group of genetic conditions caused by an aberration in a sex chromosome in which there is missing or extra sex chromosome material. These numerical changes in the chromosomes interfere with normal sexual development. It has been estimated that SCAs occur in one of every 400 live births. The impact of fetal SCA on general health including psychosocial development varies. These aneuploidies are typically diagnosed postnatally with many individuals going undiagnosed until they seek medical care for fertility problems. SCAs are also sometimes diagnosed as a result of invasive karyotype testing of pregnant women at high risk for Down syndrome. NIPT is being explored as a screening tool to identify common sex chromosome aneuploidies including, but not limited to the following:
Currently, there is less peer-reviewed published evidence on the diagnostic performance of NIPTs for detecting fetal SCAs. Limited data indicate that NIPT has a lower accuracy for SCA than for trisomies 21 and 18. The sensitivity for sex chromosome abnormalities average 91% and have a sensitivity of greater than 99%, however, the values depend on the particular condition identified. The positive and negative predictive values for SCAs are dependent upon the particular condition identified. In general, the positive predictive value ranges from 20-40% for most of these conditions (ACOG, 2015).
Gil and colleagues (2015) conducted a systematic review and meta-analysis to review clinical validation or implementation studies of maternal blood cell-free DNA analysis and define the performance of screening for fetal trisomies 21, 18 and 13 and sex chromosome aneuploidies. Searches of the peer-reviewed literature between January 2011 and January 2015 yielded a total of 37 studies, however, only 28 of these studies reported on sex chromosome aneuploidy testing. Sixteen of the 28 studies focused on the detection of monosomy X (Turners syndrome). Of the 177 singleton pregnancies with fetal monosomy X, the detection rate fluctuated between 66.7% and 100% and the false-positive rate ranged between 0% and 0.52%. The authors reported the pooled weighted detection rate was 90.3% (95% CI, 85.7-94.2%), and the false-positive rate was 0.23% (95% CI, 0.14-0.34%). Of the remaining 12 studies that focused on the performance of sex chromosome abnormalities other than monosomy X, the pooled detection rate was 93.0% (95% CI, 85.8-97.8% and the false-positive rate was 0.14% (95% CI, 0.06-0.24%). Limitations of the study include but are not limited to its small sample sizes and not clearly defining the participant's risk category.
Wang and colleagues (2015) studied the concordance of NIPT results among 109 consecutive cases with positive or negative NIPT results and compared those findings with the cytogenetic prenatal and/or postnatal karyotype results. A total of 16 of these cases were tested for fetal sex chromosome aneuploidies. Of these 16 cases, the true positive rate was 38% (6/16 cases), and the false positive rate was 62% (10/16 cases). The authors concluded that there is a need for a careful interpretation of non-invasive prenatal testing results. Limitations of this study include its small sample size and the fact that testing was limited to one of several main laboratories performing NIPT in the U.S., all of which use different algorithms or methodologies.
The joint position statement of the European Society of Human Genetics (ESHG) and the Social Issues Committee of the American Society of Human Genetics (ASHG) recommends against expanding NIPT-based prenatal screening to also report on SCAs (Dondorp, 2015).
At the time of this review, no direct comparative evidence was identified that demonstrated cell-free fetal DNA‒based screening for SCA resulted in a change in clinical management. It is unclear what effect cell-free DNA-based screening for sex chromosome aneuploidies will have on net health outcomes.
NIPT using cell-free DNA is being researched as a tool to screen for microdeletions. Microdeletions (also referred to as submicroscopic deletions) are chromosomal deletions that are too small to be detected by conventional cytogenetic methods or microscopy. Microdeletions, in conjunction with microduplications, are collectively known as copy number variations (CNVs). CNVs can lead to disease development when the change in copy number of a dose-sensitive gene or genes disturbs the ability of the gene(s) to function and effects the volume of protein produced.
Several genomic disorders associated with microdeletion have been identified. Microdeletion syndromes have distinctive and, in many cases, serious clinical features, including cardiac anomalies, immune deficiency, palatal defects, and cognitive delay. While some microdeletions are inherited, other occur de novo. Microdeletion syndromes include, but are not limited to the following:
The joint position statement of the European Society of Human Genetics (ESHG) and the Social Issues Committee of the American Society of Human Genetics (ASHG) recommends against NIPT-based screening for chromosomal microdeletion syndromes and states the following:
Concerns have been raised that this expansion of the screening offer is based on proof of principle rather than validation studies, and that with the rarity of most of these microdeletion syndromes, the PPV is expected to be low. Multiple false positives as a result of screening for microdeletions will undermine the main achievement of NIPT in the context of prenatal screening: the significant reduction of the invasive testing rate. Depending on the resolution used for expanded NIPT, more of the recently identified smaller microdeletion (and duplication) syndromes may also be detected. Many of these are associated with generally milder phenotypes, whereas some may even be present in healthy individuals. With higher resolutions, variants may also be found of which the clinical significance is still unknown. Screening for these conditions and subsequent follow-up testing (also of the parents) will lead to information and counseling challenges, as well as burdening pregnant women or couples with difficult decision making (Dondorp, 2015).
The ACOG Committee Opinion on the use of cell-free DNA screening for fetal aneuploidy states the following:
Microdeletion syndromes occur sporadically or are due to other genetic mechanisms. Screening for these microdeletions has not been validated in clinical studies and the true sensitivity and specificity of this screening test is uncertain. Routine cell-free DNA screening for microdeletion syndromes should not be performed (ACOG, 2015).
The clinical implications of prenatal testing for microdeletions are not clearly defined. It has not yet been determined whether prenatal diagnosis is appropriate given the inherent complexity of accurately predicting the phenotype for the numerous of microdeletion syndromes.
For all pregnancies, the baseline risk of some type of birth defect occurring is 3 to 4 percent. The severity of such defects varies greatly, and is a reflection of the wide range of possible inherited mutations or genetic variants. Spontaneous mutations can rise in the gametes, embryo or fetus or be the result of epigenetic alterations or environmental influences. Maternal factors that increase the risk of having a child with a congenital anomaly or genetic condition include advanced maternal age (over the age of 35 at the time of delivery), diabetes, obesity and exposure to teratogenic factors such as viral infections and alcohol (Bodurtha, 2012).
Adrenoleukodystrohy is an x-linked genetic disorder that occurs primarily in males and affects the nervous system and the adrenal glands. In this disorder, the myelin is prone to deterioration which reduces the ability of the nerves to relay information to the brain. Additionally, damage to the adrenal cortex results in a shortage of certain hormones (adrenocortical insufficiency). Adrenocortical insufficiency may cause skin changes, weakness, weight loss, vomiting, and coma. Adrenoleukodystrohy is typically divided into three categories:
Children with the cerebral form of X-linked adrenoleukodystrophy may experience learning and behavioral problems that typically begin between 4 and 10 years of age. Over time the symptoms worsen, and these children may have difficulty reading, writing, comprehending written material and understanding speech. Additional signs and symptoms of the cerebral form include vision problems, difficulty swallowing, poor coordination, aggressive behavior, and impaired adrenal gland function. The rate at which this disorder progresses varies but can be rapid, often resulting in total disability within a few years (Genetics Home Reference, X-linked adrenoleukodystrophy, 2016).
Signs and symptoms of the adrenomyeloneuropathy type are typically of late onset and may appear between early adulthood and middle age. Affected individuals may experience progressive stiffness and paraparesis, urinary and genital tract disorders as well as changes in behavior and cognitive ability. Most individuals with the adrenomyeloneuropathy type also have adrenocortical insufficiency. In severely affected individuals, damage to the brain and nervous system may lead to early death (Genetics Home Reference, X-linked adrenoleukodystrophy, 2016).
Individuals with X-linked adrenoleukodystrophy whose only symptom is adrenocortical insufficiency are characterized as having the Addison disease only form. In these individuals, adrenocortical insufficiency may begin at any time between childhood and adulthood. However, these affected individuals typically develop the additional features of the adrenomyeloneuropathy type by the time they reach middle age. The life expectancy of individuals with this form varies depending on the severity of the signs and symptoms (Genetics Home Reference, X-linked adrenoleukodystrophy, 2016).
Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia is a term used to collectively refer to group of recessively inherited disorders of cortisol production, which, when manifested in the classical form, result in virilisation of female fetuses. Treating the expectant mother with dexamethasone has been recommended in high-risk pregnancies to minimize the risk of virilising the female genitalia of affected fetuses. In order to be effective, dexamethasone treatment must be implemented early during the pregnancy, ideally at 6 weeks and certainly prior to 9 weeks gestation, when the fetal adrenal cortex begins to secrete androgens. Utilizing this approach, 7 of 8 high-risk pregnancies are treated unnecessarily, prior to establishing the fetal gender or the confirmed diagnosis of a genetically affected pregnancy. Dexamethasone administered during the antenatal is still considered by some experts as an appropriate treatment to offer for fetal CAH because it reduces female virilisation, helps avoid the need for postnatal genital reconstructive surgery and precludes the emotional distress experienced by parents having a child with ambiguous genitalia. However, prenatal treatment with dexamethasone is associated with substantial maternal side effects, including edema, weight gain, mood change, sleep disturbance, acne and the development of striae. Concerns regarding the fetal impact of antenatal dexamethasone exposure are related to potential long-term sequelae of glucocorticoid exposure in utero, including effects on growth, metabolism, cognitive function and disturbance of the hypothalamic–pituitary–adrenal (HPA) axis. Due to the potential adverse effects of high-dose dexamethasone on the mother and the long-term neurocognitive and metabolic effects on the fetus, researchers are exploring gender determination using non-invasive prenatal testing in early pregnancy prior to the commencement of dexamethasone treatment (Heland, 2016).
Duchenne Muscular Dystrophy
Duchenne Muscular Dystrophy is an x-linked recessive genetic disorder characterized by progressive muscle atrophy. This form of muscular dystrophy primarily affects the skeletal and cardiac muscles and occurs almost exclusively in males. In this condition, muscle weakness tends to appear in early childhood and worsen rapidly. Affected children may demonstrate delayed motor skills, such as sitting, standing, walking and are usually wheelchair-dependent by adolescence. The onset of cardiomyopathy typically begins in adolescence (Genetics Home Reference, Duchenne and Becker muscular dystrophy, 2016).
Hemophilia is a group of hereditary genetic disorders that impair the blood clotting process. Hemophilia A is caused by changes in the F8 gene while hemophilia B is caused by mutations in the F9 gene. Another form of hemophilia is acquired after birth and is not an inherited disorder. Individuals with hemophilia experience prolonged bleeding or oozing following an injury or a surgical procedure. In the more severe cases, continuous bleeding occurs after minor trauma or in the absence of injury (spontaneous bleeding). Hemophilia may result in serious complications as a result of bleeding into the joints, muscles, brain, or other internal organs. In milder forms of the condition, the individual does not necessarily experience spontaneous bleeding, and the condition may not be diagnosed until abnormal bleeding occurs following surgery or a serious injury. (Genetics Home Reference, Hemophilia, 2016)
Trisomy 21 (Down syndrome)
Down syndrome is the result of 1 of 3 types of abnormal cell division involving chromosome 21. Any of these 3 abnormal types of cell division result in extra genetic material from chromosome 21, which is responsible for the characteristic features and developmental problems of Down syndrome. The genetic variations that can cause Down syndrome include:
Most of the time, Down syndrome is not inherited, but occurs de novo ("from the beginning"), as a result of a mistake in cell division during the development of the egg, sperm or embryo. Translocation Down syndrome is the only form of the disorder that can be inherited. When translocations are inherited, the parent (father or mother) is a balanced carrier of the translocation (the parent has some rearranged genetic material, but no extra genetic material). Balanced carriers exhibit no signs or symptoms of Down syndrome, but they can pass the translocation on to their offspring.
Trisomy 21 or Down syndrome is the most common chromosome abnormality in humans with an incidence of approximately 1 in 800 live births (Driscoll, 2009). Although the risk of trisomy 21 increases with maternal age, most trisomy 21 pregnancies occur in women younger than age 35. Screening for trisomy 21 during the first trimester involves a combination of maternal serum beta human chorionic gonadotropin (β-HCG), maternal serum pregnancy-associated plasma protein-A (PAPP-A) and ultrasound measurement of nuchal translucency. These markers along with maternal age are used to estimate the risk of a trisomy 21 fetus. Sensitivity with this screening panel is reported to be 85% with a false positive rate of 5%. Invasive testing to diagnose trisomy 21 prenatally includes amniocentesis or CVS to obtain fetal cells for karyotyping. Both procedures involve risk of pregnancy loss and are generally performed only on women whose risk of trisomy 21 based on the biochemical screening profile and/or ultrasound findings is greater than the risk of pregnancy loss from an invasive diagnostic test (approximately 0.5% for amniocentesis and 0.5-1.0% for CVS).
Monosomy X (Turner syndrome, monosomy XO)
Turner syndrome results when one normal X (sex) chromosome is present in a female's cells and the other sex chromosome is missing or structurally altered. The missing genetic material affects the development of the female both before and after birth. The most common feature of Turner syndrome is short stature, which generally becomes evident by age 5. An early loss of ovarian function is also very common. Affected girls may not undergo puberty unless they receive hormone therapy and most are infertile. A small percentage of females with Turner syndrome retain normal ovarian function through young adulthood. Other features of Turner syndrome include extra folds of skin on the neck (webbed neck), a low hairline at the back of the neck, lymphedema of the extremities, skeletal abnormalities, or kidney problems. One third to one half of individuals with Turner syndrome are born with a heart defect which may become life-threatening. Most females with Turner syndrome have normal intelligence; however, developmental delays, nonverbal learning disabilities, and behavioral problems are possible, although these characteristics vary among affected individuals. Turner syndrome occurs in approximately 1 in 2500 newborn girls worldwide, but it is much more common among pregnancies that do not survive to term (miscarriages and stillbirths).
Trisomy X (Triple X syndrome)
Trisomy X (also known as Triple X syndrome) occurs in approximately 1 in 1000 newborn girls with 5 to 10 females being born with this condition in the United States each day. Females with the Triple X syndrome have an extra (3) X chromosome instead of the usual pair (2) of sex chromosomes. Although this condition is genetic, it is generally not inherited. Instead, it is usually the result of either the mother's egg or the father's sperm not forming correctly, resulting in an extra X chromosome. Females with this condition may be taller than average, but may have no other unusual physical features. Most females with triple X syndrome reach normal sexual development and are able to conceive children. Triple X syndrome is associated with an increased risk of developmental delay of speech and language skills and learning disabilities. Delayed motor skills (sitting and walking) development, hypotonia, as well as behavioral and emotional difficulties are also possible. The severity of these conditions varies widely among affected individuals. Seizure disorder or kidney abnormalities may occur in approximately 10% of the affected females.
Trisomy 13 (Patau syndrome) is the least common (occurring in approximately 1 in 16,000 newborns) and 1 of the most severe of the autosomal trisomies. Many of the clinical features widely vary; however, severe mental deficiency is a consistent feature in children born with Patau syndrome. Brain malformation, polydactyly, rocker-bottom feet, facial clefting, neural tube defects, missing ribs and heart defects are also generally present. Patau syndrome is frequently recognized at birth by the presence of structural birth defects and poor neurologic performance. Median survival age for infants with Patau syndrome is 2.5 days, with only 1 in 20 infants surviving longer than 6 months. Although the exact cause of Patau syndrome has not been identified, a significant association is recognized between Patau syndrome and increased maternal age.
Trisomy 18 (Edwards syndrome) occurs in approximately 1 in 5000 live births. Individuals with trisomy 18 often have intrauterine growth retardation (slow growth prior to birth) and a low birth weight. Affected individuals may have heart defects (atrial septal defect, patent ductus arteriosus, or ventricular septal defect) and abnormalities of other organs that develop before birth. Other features of trisomy 18 include camptodactyly with overlapping fingers, rocker-bottom feet, low-set ears, mental deficiency, microcephaly, micrognathia, undescended testicles, underdeveloped fingernails, and pectus carinatum. Due to the presence of several life-threatening medical problems, many individuals with trisomy 18 die before birth or within their first month of life. Only 5-10% of affected individuals live past their first year, and those that do survive often have severe intellectual disability. Like trisomy 13, the development of trisomy 18 is also associated with advanced maternal age.
Turner syndrome (monosomy X or 45, X)
Turner syndrome is an X chromosome disorder that generally affects females. The most common feature of Turner syndrome is short stature, which becomes evident by 5 years of age. Ovarian hypofunction or premature ovarian failure is very common. Many females with Turner syndrome do not undergo puberty unless they receive hormone therapy, and are infertile. A small percentage of females retain normal ovarian function through young adulthood. Approximately 30% of females with Turner syndrome have extra folds of skin on the neck, a low hairline at the back of the neck, lymphedema of the hands and feet, kidney problems or skeletal abnormalities. Approximately one-third to one-half of individuals with Turner syndrome are born with a heart defect, such as a narrowing of the large artery leaving the heart (coarctation of the aorta) or abnormalities of the valve that connects the aorta with the heart (the aortic valve). Complications associated with these heart defects can be life-threatening.
There are currently several commercially available laboratory developed tests which analyze circulating cell-free DNA to identify increased representation of various chromosomes. These tests include but are not necessarily limited to the following:
While most if not all laboratories currently offer cell-free DNA screening for aneuploidy include trisomies 13, 18 and 21 as part of a standard panel, the approach to sex chromosomes and other chromosome abnormalities vary. Some laboratories offer routine microdeletion, sex chromosome and rare trisomy (for example trisomy 22 or trisomy 16) assessment, whereas others require that sex chromosome and other assessments be specifically requested in order for those results to be reported (ACOG, 2015).
According to the National Society of Genetic Counselors (NSGC), genetic counseling is the process of assisting individuals to understand and adapt to the medical, psychological and familial ramifications of a genetic disease. This process typically includes the guidance of a specially trained professional who:
Adrenal cortex: The outer layer of the adrenal gland(s).
Aneuploidy: A condition where there are either fewer or more than the normal number of chromosomes present in cells of a person's body.
Camptodactyly: Permanently closed (flexed) fingers.
Chorionic villus sampling: A prenatal test to detect birth defects which involves retrieval and examination of tissue from the chorionic villi (placenta).
Circulating cell-free fetal DNA (ccffDNA): The result of the breakdown of fetal cells (mostly placental) which clears from the maternal system within hours.
Euploidy: The state of having a balanced set of chromosomes.
Fetal fraction: A measurement of the amount of cell-free DNA material in the maternal blood that is of fetal origin.
Holoprosencephaly: Failure of the forebrain to divide into lobes or hemispheres.
Hypotonia: Weak muscle tone.
Microcephaly: A small head.
Micrognathia: A small jaw.
Monosomy: The absence of one chromosome of the usual pair to (two) chromosomes.
Monosomy X: A congenital disorder caused by the absence of one X sex chromosome (the individual has only one X sex chromosome rather than the usual pair [either two Xs or one X and one Y sex chromosome]); also called Turner syndrome and monosomy XO.
Myelin: The fatty covering that insulates nerves in the spinal cord and brain.
Pectus carinatum: An unusual shaped chest.
Polydactyly: Extra fingers or toes.
Robertsonian translocations: Chromosomal rearrangement in humans that occurs in five acrocentric (long and short arm) chromosome pairs (13, 14, 15, 21, and 22). These translocations are also called "whole-arm" or centric-fusion translocations. During a Robertsonian translocation, the chromosomes break at their centromeres and the long arms fuse to form a single chromosome with a single centromere. The short arms also join, but usually contain nonessential genes and are usually lost within a few cell divisions.
Rocker-bottom feet: A rigid flatfoot deformity.
Sex chromosome aneuploidy: A group of genetic conditions caused by an abnormal sex chromosome aberration in which there is missing or extra sex chromosome material.
Trisomy: The presence of three chromosomes, rather than the usual pair of (two) chromosomes.
Trisomy X: A congenital disorder caused by having an extra copy of chromosome X; also called Triple X syndrome.
Trisomy 13: A congenital disorder caused by having an extra copy of chromosome 13; also called Patau syndrome.
Trisomy 18: A congenital disorder caused by having an extra copy of chromosome 18; also called Edwards syndrome.
Trisomy 21: A congenital disorder caused by having an extra copy of chromosome 21; also called Down syndrome.
Virilisation: The development of male physical characteristics (such as body hair, deep voice and muscle bulk) in a female or at an unusually early age in a boy, typically due to excess androgen production.
The following codes for treatments and procedures applicable to this document are included below for informational purposes. Inclusion or exclusion of a procedure, diagnosis or device code(s) does not constitute or imply member coverage or provider reimbursement policy. Please refer to the member's contract benefits in effect at the time of service to determine coverage or non-coverage of these services as it applies to an individual member.
When services may be Medically Necessary when criteria are met:
|81420||Fetal chromosomal aneuploidy (eg, trisomy 21, monosomy X) genomic sequence analysis panel, circulating cell-free fetal DNA in maternal blood, must include analysis of chromosomes 13, 18, and 21|
|81479||Unlisted molecular pathology procedure [when specified as cell-free fetal DNA-based prenatal testing for fetal aneuploidy or sex determination]|
|81507||Fetal aneuploidy (trisomy 21, 18, and 13) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy (Harmony Prenatal Test, Ariosa Diagnostics)|
|0009M||Fetal aneuploidy (trisomy 21, and 18) DNA sequence analysis of selected regions using maternal plasma, algorithm reported as a risk score for each trisomy (VisibiliT™, Sequenom Center for Molecular Medicine, LLC)|
|81599||Unlisted multianalyte assay with algorithmic analysis [when specified as cell-free fetal DNA-based prenatal testing involving multianalyte assays and an algorithmic analysis for fetal aneuploidy or sex determination]|
|All other pregnancy diagnoses not listed as investigational and not medically necessary|
When services are Not Medically Necessary:
For the procedure codes listed above for fetal sex determination when criteria are not met, or when the code describes a procedure indicated in the Position Statement section as not medically necessary.
When services are Investigational and Not Medically Necessary:
For the procedure codes listed above for the following diagnosis codes, or when the code describes a procedure indicated in the Position Statement section as investigational and not medically necessary.
|O31.00X0-O31.8X99||Complications specific to multiple gestation|
When services are also Investigational and Not Medically Necessary:
|81422||Fetal chromosomal microdeletion(s) genomic sequence analysis (eg, DiGeorge syndrome, Cri-du-chat syndrome), circulating cell-free fetal DNA in maternal blood|
|81479||Unlisted molecular pathology procedure [when specified as cell-free fetal DNA-based prenatal testing of the genome for other chromosomal abnormalities]|
Peer Reviewed Publications:
Government Agency, Medical Society, and Other Authoritative Publications:
|Websites for Additional Information|
Fetal sex chromosome aneuploidy
Fetal sex determination
Harmony Prenatal Test
Massively parallel DNA sequencing
Panorama Prenatal Test
Triple X syndrome
verifi Prenatal Test
VisibiliT Prenatal Test
The use of specific product names is illustrative only. It is not intended to be a recommendation of one product over another, and is not intended to represent a complete listing of all products available.
|Reviewed||08/03/2017||Medical Policy & Technology Assessment Committee (MPTAC) review. Updated Rationale, References, Index and History sections.|
|Revised||05/04/2017||MPTAC review. Updated the medically necessary statements to include criteria for genetic counseling. Updated Coding, References and History sections.|
|01/01/2017||Updated Coding section to include 01/01/2017 CPT changes.|
|Revised||08/04/2016||MPTAC review. Title changed to "Cell-Free Fetal DNA-Based Prenatal Testing". Scope of document expanded to address testing for fetal sex determination, microdeletions and sex chromosome aneuploidies. Updated Description/Scope, Rationale, Background/Overview, References, Index and History section. Updated Coding section and removed ICD-9 codes.|
|Revised||09/18/2015||MPTAC review. Revised Position Statement to consider cell-free fetal DNA-based prenatal screening for fetal aneuploidy (trisomy 13, 18, and 21) as medically necessary for women with a current single gestation pregnancy. Updated the Description, Rationale, References, Websites for Additional Information and Index sections.|
|Revised||08/06/2015||MPTAC review. Position statement revised to indicate that cell-free DNA-based prenatal screening for fetal aneuploidy (trisomy 13, 18 and 21) is considered medically necessary when the individual is carrying a single gestation, regardless of the individual's risk status for fetal aneuploidy and provided the individual understands the limitations and benefits of the screening paradigm. Revised the investigational and not medically necessary statement. Updated review date, Rationale, Coding and References sections.|
|07/01/2015||Updated Coding section with 07/01/2015 CPT changes.|
|Revised||02/05/2015||MPTAC review. Added "fetal" to investigational and not medically necessary position statement. Updated Background/Overview, Rationale, and References sections.|
|01/01/2015||Updated Coding section with 01/01/2015 CPT changes.|
|Reviewed||02/13/2014||MPTAC review. Updated Rationale, Background/Overview and References sections.|
|01/01/2014||Updated Coding section with 01/01/2014 CPT changes; removed 0005M deleted 12/31/2013.|
|07/01/2013||Updated Coding section with 07/01/2013 CPT changes.|
|Reviewed||02/14/2013||MPTAC review. Updated review date, References and History sections.|
|Revised||12/17/2012||MPTAC review. Title changed to "Cell-free DNA-Based Prenatal Screening for Fetal Aneuploidy." Position changed to consider cell-free DNA-based prenatal screening for fetal aneuploidy medically necessary when criteria are met. Investigational and not medically necessary statement revised to stipulate testing is not covered in women not at high risk for fetal aneuploidy and for women with a current multiple gestation pregnancy. Rationale, Background/Overview, Definitions, References and History sections updated. Updated Coding section to include 01/01/2013 CPT changes.|
|Revised||08/09/2012||Medical Policy & Technology Assessment Committee (MPTAC) review. Expanded scope of document to address Trisomies 13, 18, 21, X and monosomy X. Title changed to "DNA-Based Noninvasive Prenatal Diagnostic Testing for Fetal Aneuploidy. Revised position statement to specifically address DNA-based noninvasive prenatal diagnostic testing for trisomies 13, 18 and X. Updated the Rationale, Background/Overview, Definitions, References, Index and History sections.|
|New||05/10/2012||MPTAC review. Initial document development.|