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Chromosomal abnormalities not currently detected by cell-free fetal DNA: a retrospective analysis at a single center

American Journal of Obstetrics and Gynecology, Volume 214, Issue 6, June 2016, Pages 729.e1 - 729.e11


Cell-free fetal DNA analysis is used as a screening test to identify pregnancies that are at risk for common autosomal and sex chromosome aneuploidies.


The purpose of this study was to investigate the chromosomal abnormalities that would not be detected by cell-free fetal DNA in a single medical center.

Study Design

This was a retrospective cohort analysis of 3182 consecutive invasive diagnostic procedures that were performed at Montefiore Medical Center’s Division of Reproductive and Medical Genetics from January 1, 2009 to August 31, 2014. All patients underwent cytogenetic analysis; one-third of the patients (1037/3182) went through chromosomal microarray analysis.


Clinically significant chromosomal abnormalities were detected in 220 of 3140 cases (7%) after we excluded multiple gestation pregnancies (n = 42). Of these 125 cases (57%) were diagnosed with the common autosomal trisomies that involved chromosomes 21, 18, and 13 and with sex chromosome aneuploidies. There were 23 mosaic karyotypes; 8 of them involved trisomy in chromosomes 21 and 13; 5 of them were sex chromosome mosaics, and 10 of them were other mosaic cases. Five cases of triploidy were detected. Additionally, 19 unbalanced chromosomal rearrangements, a rare autosomal trisomy, and 47 clinically significant findings on chromosomal microarray analysis were diagnosed. Based on the published detection rates of cell-free fetal DNA testing and considering the “no-results” rate, we calculated that 99 of 220 chromosomal changes (45%) could not have been detected by cell-free fetal DNA testing: 16 of the 125 common aneuploidies and sex chromosome aneuploidies, 1 of the 5 triploidy cases, 15 of the 23 mosaic cases, all cases of unbalanced chromosomal rearrangements (n = 19), rare autosomal trisomy (n = 1), and 47 clinically significant chromosomal microarray abnormalities.


Current cell-free DNA testing could not detect up to one-half of the clinically significant chromosomal abnormalities that were found, which included clinically significant chromosomal microarray abnormalities. Among the 99 abnormal karyotypes that were not identified by cell-free DNA screening, 79% were from women with abnormal screening or abnormal ultrasound finding; 21% were from women who underwent invasive testing simply for advanced maternal age/concern, with no other risk factors or ultrasound findings. This information highlights the limitations of cell-free DNA screening and the importance of counseling patients about all prenatal screening and diagnostic procedures and about the added gain of invasive testing with karyotype and microarray.

Key words: cell-free fetal DNA, detection rate, diagnostic tests.

Cell-free fetal DNA (cffDNA) testing is a screening test that shows unsurpassed sensitivity for the detection of trisomy 21, both in the high-risk and the low-risk population.1, 2, 3, 4, 5, 6, 7, and 8 CffDNA testing also shows good results in the identification of pregnancies that are at risk for other common autosomal aneuploidies (trisomy 18 and trisomy 13).1, 3, 5, 9, 10, 11, and 12 Detection of sex chromosome abnormalities is also improving, and recent studies have shown promising results.3 and 13 Detection rates (DRs) for mosaics currently are undetermined, and the detection of triploidy with cffDNA depends on the method that is used and on whether it is a diandric or a digynic triploidy.14, 15, and 16 Many national organizations have set guidelines for the use of cffDNA for aneuploidy screening with a collective conclusion that patients who are at increased risk for aneuploidy can be offered cffDNA screening with appropriate pretest counseling.17, 18, 19, 20, 21, 22, and 23

Amniocentesis and chorionic villus sampling (CVS) are invasive diagnostic procedures for the investigation of fetal chromosomal and subchromosomal abnormalities; both carry a risk for miscarriage. According to the recent metaanalysis,24 the weighted pooled procedure-related risks of miscarriage for amniocentesis and CVS were 0.11% (95% CI, –0.04 to 0.26%) and 0.22% (95% CI, –0.71 to 1.16%), respectively.

The National Institutes of Health (NIH)–sponsored clinical trial investigated the accuracy of fetal diagnosis by comparing metaphase karyotype and chromosomal microarray analysis (CMA) and showed that there is an increase in the detection of clinically significant CMA abnormalities, even when the metaphase karyotype was normal.25

It was reported previously that 17.4% of pregnancies with a positive quadruple test result had karyotype other than the common trisomies (trisomy 21 or trisomy 18/13).26 Additionally, among patients who underwent invasive prenatal diagnosis because of a positive first-trimester screening (FTS), nearly 30% of the patients were found to have a chromosomal abnormality on karyotype other than the common trisomies27; however, CMA abnormalities were not included in these studies.

It is of concern that the use of cffDNA to rule out trisomies 21,18, or 13 after a positive first- or second-trimester screening test might result in a diminution in the chromosomal abnormalities (microscopic and submicroscopic) that can be detected with the use of the current invasive procedures.

We aimed to ascertain the percentage of chromosomal abnormalities that would be missed if only cffDNA testing was performed in an underserved, high-risk population.

Materials and Methods

We report a retrospective cohort analysis of 3182 consecutive amniocentesis and CVS procedures performed at Montefiore Medical Center’s Division of Reproductive Genetics from January 1, 2009 (the introduction of CMA in our center) to July 31, 2014.

All women who are treated at our medical center are offered a traditional screening test: FTS (nuchal translucency [NT] and analytes) when they initiate prenatal care early in pregnancy or quadruple screening (analytes alone) for patients who need prenatal care past the first trimester and up to 21 weeks gestation. A mid-trimester detailed anatomy scan is offered to all patients. High-risk patients and the patients who are interested in invasive testing are referred for genetic counseling.28 If a patient chooses to have a diagnostic test, a CVS or amniocentesis (10 to 13 + 6/7 and 16-23 weeks gestation, respectively) is performed. Invasive procedures are performed on-site at our center; a standard metaphase cytogenetic analysis of cells that are obtained by amniocentesis or CVS is performed in one of the authorized diagnostic laboratories routinely used by our institute. Array-based comparative genomic hybridization (aCGH) has been used at our center since 2009 (2009-2010 as part of an NIH array study that used mainly oligonucleotide probes25 and since 2010 have used a single nucleotide polymorphism [SNP] platform for most patients and oligonucleotide platform for the remainder of patients, depending on insurance coverage and referent laboratory). Until 2013, aCGH was offered only to high-risk patients who were having an invasive procedure (high risk includes advanced maternal age [AMA] and maternal age adjusted risk after screen positive test, women who had a previous fetus/child affected by autosomal trisomy, structural anomalies identified by ultrasonography and parental carrier of chromosomal rearrangement28). Beginning in 2014, aCGH was offered to all patients who would undergo an invasive procedure as per the American College of Obstetricians and Gynecologist recommendation.29

All results were recorded in the patient’s electronic medical record and the department’s log books by board-certified genetic counselors and were reviewed by medical geneticists. Results were categorized into common aneuploidies (involving trisomies in chromosomes 21, 18, and 13), sex chromosome aneuploidies (monosomy X, XXX, Klinefelter syndrome and XYY syndrome), triploidy, unbalanced chromosomal rearrangements (translocation, inversion and deletion/duplication), mosaics, and CMA abnormalities.

Statistical analysis

The Student t test and Pearson’s chi-square test were used to evaluate the statistical significance of the comparison of the indication for procedure in the normal and abnormal results groups. A probability value of <.05 was considered to indicate statistical significance.

Calculation of detectability by cffDNA

We used the following weighted pooled DR and false-positive rates (FPR), based on the recent metaanalysis of studies of maternal peripheral blood cffDNA analysis30: For trisomy 21, 99.2% DR (95% CI, 98.5–99.6%) with 0.09% FPR (95% CI, 0.05–0.14%); for trisomy 18, 96.3% DR (95% CI, 94.3–97.9%) with 0.13% FPR (95% CI, 0.07–0.20%); for trisomy 13, 91.0% DR (95% CI, 85.0–95.6%) with 0.13% FPR (95% CI, 0.05–0.26%); for monosomy X, 90.3% DR (95% CI, 85.7–94.2%) with 0.23% FPR (95% CI, 0.14–0.34%); for sex chromosome aneuploidies other than monosomy X, 93.0% DR (95% CI, 85.8–97.8%) with 0.14% FPR (95% CI, 0.06–0.24%).

The DR of triploidy was calculated based on available publications at this time; aiming to identify fetal triploidy using cffDNA. Nicolaides et al14 showed the correct identification of 4 of 4 diandric triploidy using a SNP-based cffDNA. That same method failed to detect 4 of 4 digynic triploidy. Others also demonstrated very low fetal fraction in digynic triploidy (fetal fraction <3%, no result reported on cffDNA)15 and correct identification of diandric triploidy with the use of SNP.16 Hence, a 100% DR for diandric triploidy with SNP-based cffDNA testing and 0% DR for digynic triploidy were assumed.

Clinical validation trials report a wide range of DRs of mosaics cases by cffDNA analysis3, 31, and 32; 3 of 3 of mosaic trisomy 21 and 1 of 1 mosaic trisomy 18 were detected by cffDNA; the DR reported for monosomy X mosaic is 2 of 7 (29%)3. CffDNA test has not been reported to detect other complex mosaics.3 Others have reported cffDNA analysis to detect only 1 of 2 cases of mosaic trisomy 21.32 Also, cffDNA could not detect mosaic trisomy 13 and mosaic trisomy 21 superimposed with mosaic T18 (trisomy 21 was detected, but the mosaic T18 was not).32 We conservatively calculated the common trisomies mosaic DR as the same as we calculated for the complete aneuploidies. This is probably an overestimation because the contribution of the fetal excess chromosome is partial; therefore, the DR is expected to be lower compared with the DR of the complete trisomies. For monosomy X mosaic, we calculated 29% DR.3 For other sex chromosome aneuploidy mosaic, there is no published DR.

To date, there are no data available for the cffDNA DR of unbalanced chromosomal rearrangements or CMA abnormalities.

Data on the no-result rate because of assay failure or low fetal fraction varies dramatically from 0.52-6.1%33 and recently 3% in the United States1 and 0.1% in China (with 2.18% required repeat blood sampling).34 In pregnancies that are complicated with chromosomal aneuploidies, the rate of no-result is increased. Pergament et al33 reported 16% of aneuploidies had no-result on cffDNA analysis. On the basis of the recent metaanalysis by Gil et al,30 we assumed that, in 6.9% of trisomies (complete and mosaic) and in 17.2% of sex chromosome aneuploidies (complete and mosaic), a no-result will be received.

The study was approved by the Albert Einstein Medical Center institutional review board (IRB Number: 2014-4252).


Between January 1, 2009, and July 31, 2014, 3182 consecutive procedures were performed at Montefiore Medical Center’s Division of Reproductive Genetics: 2514 amniocenteses and 668 CVSs. Forty-two procedures were excluded because of multiple gestations: 33 procedures were done for 20 twin pairs (of them 6 were monochorionic) and 9 procedures were done for 2 triplet and 1 quadruplet pregnancies.

All 3140 samples had standard karyotype; 1037 samples also had aCGH performed. Maternal age ranged from 15-55 years (median, 38 years). Indications for the procedure in the study cohort divided by normal and abnormal results groups are listed in Table 1.

Table 1 Indications for invasive procedure in the normal results group vs abnormal result group

Indication Normal results groupa (n = 2920), n (%) Clinically significant abnormal result group (n = 220), n (%) P value
Advanced maternal age 858 (29.4) 30 (13.6) <.0001
Increased nuchal translucency 144 (4.9) 47 (21.4) <.0001
Abnormal ultrasound finding 501 (17.2) 67 (30.1) <.0001
Known parental chromosomal rearrangement carrier status 219 (7.5) 4 (1.8) <.01
Previous effected pregnancy 105 (3.6) 3 (1.4) .08
Maternal concern 169 (5.8) 2 (0.9) <.01
Elevated alpha fetoprotein on second trimester screen or suspected neural tube defect 95 (3.2) 3 (1.4) .12
Abnormal first trimester screen 333 (11.4) 33 (15) .16
Abnormal quadruple screen 452 (15.5) 21 (9.5) <.05
Abnormal cell-free fetal DNA results 4 (0.1) 8 (3.5) <.0001
Other 33 (1.1) 2 (0.9)
Isoimmunization 7 (0.2) 0 .46

aIncludes failed results.

Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.

Chromosomal abnormalities were determined by karyotype and aCGH. Of the 3140 standard metaphase cytogenetic analyses, chromosomal changes were detected on 208 karyotypes. Of them, there were 97 cases of common autosomal aneuploidies (involving chromosomes 21, 18, 13), 1 case of trisomy 16 on CVS, 28 cases of sex chromosome aneuploidies (21 monosomy X and 7 sex chromosome trisomies), 19 unbalanced rearrangements, and 5 triploidy cases. Twenty-three mosaic karyotypes were detected: 6 cases of trisomy 21, 2 cases of trisomy 13, 5 cases that involved sex chromosomes, and 10 other rare complex mosaic cases (Table 2). Of note, the CVS for the case of trisomy 16 was performed because of positive FTS. The patient did not have CMA; however, the fetus had complex cardiac and structural anomalies.

Table 2 Calculated detection rate of cell-free fetal DNA for invasive procedure result

Abnormalities on karyotype and chromosomal microarray analysis (n = 249) Detected, n Cell-free fetal DNA
Detection rate, %30 Failure rate, %30 Calculated detection rate in the cohort
Any autosomal aneuploidy 98
 Trisomy 21 61 99.2 6.9 61X0.992X0.931=56.3
 Trisomy 18 32 96.3 6.9 32X0.963X0.931=28.7
 Trisomy 13 4 91.0 6.9 4X0.91X0.931=3.4
 Other autosomal trisomy (trisomy 16) 1 NA 0/1
Any sex chromosome aneuploidy 28
 45X 21 90.3 17.2 21X0.903X0.828=15.7
47XXX/47XXY/47XYY 1/3/3, respectively 93.0 17.2 7X0.93X0.828=5.4
Any structural rearrangement 43
 Balanced 24 NA Not investigated in our cohort
 Unbalanced 19 NA 0/19
Triploidy 5 100, Diandric using single nucleotide polymorphism technology 0, Diandric and 100 digynic triploidy14, 15, and 16 4/5
All mosaic 28
 Common autosomal aneuploidy mosaic 8 99.2a 6.9 8X0.992X0.931=7.4
 Other autosomal aneuploidy mosaic 4 NA NA 0/4
 Sex chromosome aneuploidy mosaic 2 monosomy X, 3 others 293
 Other complex mosaics 6 NA NA 0/6
 Confined placental mosaicismb (involving chromosomes 2, 10, 16, 11, 20) 5 NA NA Not investigated in our cohort
Submicroscopic chromosomal abnormalities on chromosomal microarray analysis 47 NA NA 0/47

aA conservative estimation based on the detection rate published for complete trisomies, because of the lack of consensus regarding the detection rate of mosaics with the use of cell-free fetal DNA analysis

bIncludes 1 case of low level mosaicism on chromosomal microarray analysis that was performed on cultural cells; the patient declined amniocentesis, and no confirmation was done.

NA, not available.

Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.

Thirty-five cases were excluded from the 208 abnormal karyotype findings; 24 cases were parentally inherited balanced chromosomal rearrangements; another 6 results were duplicated; they were first noted on CVS (included in the study cohort) and later confirmed on amniocentesis (not included in the study). In addition, another 5 results were considered confined placental mosaics. All confined placental mosaics were tested for uniparental disomy (UPD). Thus, 173 of our 3140 cases (5.5%) were considered to have clinically significant chromosomal abnormalities that were found on karyotype (Figure 1).


Figure 1 Chromosomal abnormalities enrollmentFlow-sheet details the enrollment of all invasive procedures and the inclusion and exclusion of abnormal karyotype findings.CPM, confined placental mosaicism; CVS, chorionic villus sampling.Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.

Array CGH testing was performed in 1037 fetuses during the research period (33% of the patients). Over one-third of the aCGH tests (401/1037; 38.7%) were performed for fetal anomalies that were detected on ultrasonography (first-trimester NT and anatomy scan). Of the 1037 CMA studies, 870 aCGH results were considered normal. There were 100 abnormal CMA results; 53 of 100 reflected the abnormal karyotype findings, and 47 of 100 were clinically significant CMA abnormalities in the presence of normal karyotype (20 cases were considered and counseled as pathogenic changes; 23 cases were considered and counseled as likely pathogenic: 2 cases of loss of heterozygosity [LOH] pathogenic and 2 cases of UPD-likely pathogenic). Forty-eight cases of variants of unknown significance were considered benign variants and thus were excluded. Additionally there were 16 cases of nonclinically significant LOH and 1 case of nonclinically significant UPD that were excluded. In 2 cases, the laboratory failed to report the CMA results (Figure 2).


Figure 2 Chromosomal microarray enrollment and outcomeFlow-sheet details the enrollment of chromosomal microarray analysis inclusion and exclusion of findings.CMA, chromosomal microarray analysis; LOH, loss of heterozygosity; UPD, uniparental disomy.Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.

Microarray tests were considered clinically significant if an Online Mendelian Inheritance in Man (OMIM)–annotated gene was recognized in the deleted/duplicated interval and/or the deletion in the region is known to have a clinical significance. LOH was considered clinically significant if it involved known OMIM genes or if ultrasound scan revealed major anomalies that could be attributed to the LOH. UPD was considered clinically significant if previously described in the literature as having potential deleterious outcome.

A total of 220 clinically significant chromosomal abnormalities (7%; 173 microscopic and 47 submicroscopic) were detected in our population (individual karyotype and CMA findings listed in the Supplemental Table).

There were 16 cases of failed results; 7 on amniocentesis (0.28%), 9 on CVS (1.3%), and 2 CMA failures (0.2%)

Table 2 summarizes the calculated predicted cffDNA DR in our cohort, based on the published weighed pooled DRs and no-result rates of cffDNA screening.30

For the common trisomies and sex chromosome aneuploidies, we calculated that 109 of 125 cases (87.5%) would be detected by cffDNA analysis. Because of lack of consensus regarding the common trisomy mosaicism, we assumed conservatively that the DR and the no-result rates for these mosaics are the same as for the common trisomies.30 We predicted that 7 of 8 cases of mosaic trisomy 21 and 13 would have been detected by cffDNA. In addition, we predicted that 1 of 2 mosaic monosomy X cases would have been detected by cffDNA.3 A total of 8 of 23 cases of mosaics would be diagnosed by cffDNA. Our center detected 4 diandric triploidies and 1 digynic triploidy. Therefore, based on the published DR of cffDNA testing by SNP, we conservatively predict that 4 of 5 triploidy would have been detected by cffDNA.14, 15, and 16

There were 20 unbalanced chromosomal rearrangements and a rare autosomal aneuploidy and 47 clinically significant CMA findings. We predict that these would not be detected by current cffDNA.

In total, we calculated that 99 of 220 chromosomal microscopic and submicroscopic changes (45%) would not be detected by cffDNA testing (Table 2).

Fetal structural anomalies and increased NT/cystic hygroma were the indication for invasive testing in 42% of the cases (42/99) with chromosomal abnormalities assigned as nondetectable by cffDNA testing. Whereas in 21% of cases (21/99) with chromosomal abnormalities that were considered nondetectable by cffDNA, the only indication was AMA or maternal concern.

Of the 366 invasive procedures that were done for positive FTS, 9% procedures (n = 33) were screen positive. An abnormal karyotype other than the common chromosomal trisomies was present in 42% of the cases (14/33). Table 3 lists the indication for the invasive procedure in each category of chromosomal abnormality.

Table 3 Chromosomal abnormalities according to the indication for the invasive procedure

Common aneuploidy (n=97), n (%) Monosomy X (n=21), n (%) Other Sex chromosome trisomies (n=7), n (%) Triploidy (n=5), n (%) Mosaic (n=23), n (%) Chromosomal microarray analysis (n=47), n (%) Chromosomal rearrangement and a rare trisomy (n=20), n (%)
Advanced maternal age 12 (12.4) 0 (0) 1 (14.3) 0 (0) 6 (26.1) 10 (21.3) 1 (5)
1st trimester screening (%)
 First trimester screening 19 (19.6) 0 (0) 0 (0) 0 (0) 5 (21.8) 8 (17) 1 (5)
 Nuchal translucency/Cystic hygroma 24 (24.7) 16 (76.2) 0 (0) 0 (0) 2 (8.7) 3 (6.4) 2 (10)
2nd trimester screening
 Quadruple 9 (9.3) 0 (0) 3 (42.9) 0 (0) 4 (17.4) 5 (10.6) 0 (0)
 Ultrasound scan 27 (27.8) 5 (23.8) 0 (0) 4 (80) 3 (13) 18 (38.3) 10 (50)
 Elevated alpha fetoprotein 0 (0) 0 (0) 1 (14.3) 0 (0) 1 (4.3) 1 (2.1) 0 (0)
Other familial indications
 Parental chromosome rearrangement 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (20)
 Previous affected pregnancy 0 (0) 0 (0) 1 (14.3) 0 (0) 0 (0) 1 (2.1) 1 (5)
Maternal concern/others 0 (0) 0 (0) 0 (0) 1 (20) 1 (4.3) 1 (2.1) 1 (5)
Positive cell-free fetal DNA 6 (6.2) 0 (0) 1 (14.3) 0 (0) 1 (4.3) 0 (0) 0 (0)
Estimation of number not detected by cell-free fetal DNA a 7 (7.2) 7 (33.3) 1 (14.3) 1 (20) 14 (60.9) 47 (100) 20 (100)

aCalculating based on published cffDNA detection rate and no-results rate and considering the cases that were detected by cffDNA in our cohort (total of 8 cases).

Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.


The use of cffDNA as a screening test for trisomy 21 and for the common chromosomal aneuploidies is increasing rapidly; however as shown in this report, trisomies that involve chromosomes 21, 18, and 13 account for only 44% of the clinically significant chromosomal abnormalities that were found in our population. The remaining findings were monosomy X (9.5%), other sex chromosome aneuploidies (3.1%), triploidy (2.3%), mosaics (10.5%), unbalanced rearrangement, and rare trisomies (9.1%). We estimated that the current cffDNA screening test could not detect 99 of 220 clinically significant microscopic and submicroscopic changes (45%) that were found in this cohort. In previous studies, it was estimated that up to 16.9% of aneuploidies are not detected by cffDNA testing.35 and 36 However, cffDNA failure rates and CMA abnormalities were not included in the calculations.35 and 36

In the California State Screening Program, the common aneuploidies accounted for 64% of all chromosomal abnormalities.26 In another population-based study from Italy, the common aneuploidies accounted for only 58% of the unbalanced chromosomal abnormalities.37 Our work supports and expands these findings because it shows that 56% of the chromosomal abnormalities are other than the common trisomies. Additionally, of the chromosomal abnormalities that were detected after positive FTS, 42% had a chromosomal abnormality other than the common trisomies. Alamillo et al27 demonstrated that 29.9% of screen positive pregnancies with an abnormal karyotype had aneuploidy other than the common aneuploidy that was indicated by the FTS. These studies did not include CMA testing in the abnormal results.26, 27, and 37 Our work stresses that, by using cffDNA testing to confirm or rule out positive FTS results, we might oversimplify the potential meaning of that positive FTS result.

CMA analysis was done for only one-third of the cohort. We found a high incidence of clinically significant CMA abnormalities 47 of 1037 (4.5%), as we previously published.38 We believe that, if CMA were performed for the entire cohort, the percentage of clinically significant CMA abnormalities would have been higher. On the other hand, the CMA abnormalities rate may not be increased vastly because the CMA group had higher rates of anomalies that were diagnosed on ultrasound scanning and positive screening tests as an indication for the invasive procedure. Of the 1037 CMA tests that were done at our center, more than one-third (401 or 38.7%) were done for fetal anomalies. It is estimated that the probability of finding a significant abnormality on prenatal CMA testing, when karyotype is normal, is 1.7% in patients ≥35 years old or in the case of a positive FTS and up to 6% when there is an abnormal ultrasound finding.25 Further analysis of data from the original copy number variant NIH trial25 revealed that fetuses with anomalies in >1 organ system had a higher frequency of other than common benign copy number variants when compared with fetuses without anomalies (13% vs 3.6%; P < .001).39 Benachi et al40 suggested that noninvasive testing should not be offered in cases of abnormal findings on ultrasound scans.

In our entire cohort, 24% of patients (759/3140) had an abnormal ultrasound finding (including increased NT/cystic hygroma and abnormalities on detailed anatomic scan) as an indication for the invasive procedure. Of the total patients who had an abnormal ultrasound finding and elected a diagnostic procedure, 15% (114/759) had an abnormal chromosomal finding. Conversely, 51.8% of patients (114/220) with chromosomal abnormality (microscopic and submicroscopic) had abnormal ultrasound findings as an indication for the invasive procedure. It is remarkable to note that, of the chromosomal abnormalities that were considered nondetectable by cffDNA, 42% (42/99) had an abnormal ultrasound scan as the indication for the invasive procedure. Our findings further confirm that patients with an anomaly on ultrasound scanning should be counseled about the possibility of false reassurance if only cffDNA testing is used. In addition, the patients with abnormal ultrasound findings should be offered CMA for any invasive procedure, with counseling regarding the higher DR of chromosomal abnormalities. Future analysis of our data and prospective investigation may include stratification of ultrasound anomalies to assess chromosomal abnormalities by organ systems.

CffDNA sensitivity, specificity, and positive and negative predictive values for the detection of microdeletion syndromes are still undetermined. The performance of SNP-based cffDNA for 22q11.2 microdeletion currently is being evaluated in an on-going large prospective multicenter study. The DR for the 8 cases of 22q11.2 deletion/duplication in our cohort could not be estimated at this time and therefore are predicted currently to be undetected by cffDNA screening.

CffDNA DRs in cases of mosaicism currently are undetermined. Because mosaics comprise 10.5% (23/220) of the abnormal karyotype findings, the poor and unpredicted DR of cffDNA for mosaics should be noted.

There is a vast range in cffDNA failure rates.1, 2, 33, and 34 Recent studies suggest that failure rates are increased in cases of chromosomal abnormalities.1 and 33 The invasive procedure failure rate in our study was 16 of 3140 (0.5%). This information adds to the data that the patient and provider must discuss before deciding on the testing approach for the pregnancy.

More than 90% (2829/3140) of the invasive procedures that were performed at our center were done for “high-risk” indications28 (ultrasound findings, AMA, known parental chromosome abnormality, previous affected pregnancy, or positive screen). It is important to note that, of the 99 patients who were diagnosed with chromosomal abnormalities that were assumed to be missed by current cffDNA testing, 21% underwent an invasive procedure solely for maternal concern or AMA. Had cffDNA been used instead of invasive testing on all of our patients, 45% of the clinically significant abnormal results would have been missed. This percentage should be investigated in a low-risk group in future studies.

Our study was conducted in a single medical center where accessibility to records and maternal and neonatal follow-up evaluation is obtained easily. In addition, genetic counseling and invasive procedures are managed by 1 division and are similar among counselors and physicians. A limitation of the study is that CMA analysis was done only for one-third of this cohort (1037/3140). Additionally more than one-third (38.7%) of the CMA analyses were done for fetal anomalies on detailed ultrasound scan. CffDNA screen is currently not the preferred or most comprehensive test in cases of structural fetal anomalies.40 Our study could be improved with larger numbers and consistent uptake and uniform reimbursement of chromosomal microarray. It is important to note that, in many centers in the United States and abroad, CMA is not offered routinely along with invasive procedures. In such locations, the abnormalities that would be missed by cffDNA are a smaller percentage of the total findings.

The data presented in this article are beneficial to both patients and providers and add to the information to be considered regarding screening and diagnostic testing options in pregnancy. Clearly, extensive counseling of patients is paramount in this era of multiple genetic testing options.


Supplement Table 1 Clinically significant microscopic and submicroscopic chromosomal abnormalities detected at our cohort

Karyotype Abnormalities seen on microarray
46XY 0.654Mb gain 8q21.13q21.2
46XX 0.814Mb duplication 16p13.11->p13.11 duplication
46XX 1.6Mb deletion 16p13.12p13.10
46XX 0.206Mb gain 16p11.2
46XY 0.029Mb deletion 2p16.3 0.844Mb gain 6p21.2p21.1
46XY 0.394Mb gain 6p22.3, 0.332Mb deletion Xp22.33
46XY 0.635Mb deletion 6q12
46XX 0.716Mb duplication 22q11.21
46XY 0.928Mb gain 20p12.2
46XY 1.061Mb deletion 17q12
46XY 1.15Mb duplication 15q26.3->26.3 257kb deletion 16p12.2-.p12.2
46XY 1.4Mb deletion 17p12
46XX 1.5Mb deletion 22q11.21
46XY 1.6Mb gain of 16p13.11.
46XX 100kb deletion 6q12
46XX 3p21.3 1106kb gain, 147kb deletion 11p15.1
46XX 139kb deletion 7p21.2
46XY 0.224Mb deletion 15q11.2
46XY 15q13.1 deletion, 2 cases
46XY 16p13.2 gain 18q22.1 duplication
46XXinv9 1p21.1 483kb deletion , 13q31.3 215kb deletion, 16q24.3 207kb duplication
46XY 1q43q44 duplication
46XX 2.93Mb duplication16P13.11->p12.3 and 1.4Mb deletion 17P12
46XX 22q 11.2 deletion
46XY 22q11.2 deletion
46XX 22q11.2 deletion
46XY 22q11.22q11.23 duplication
46XY 22q13.33 duplication
46XX 283kb duplication Xp22.12->p22.12
46XX 424kb deletion 22q11.2
46XX 6.15Mb deletion 10q
46XX 681kb deletion 15q24.1
46XX 731kb 22q11.21 gain
46XX 761kb duplication Xp22.11
46XY 791kb deletion 9q34.11 pat
46XX 85.1.6kb duplication 18p11.31
46XY 865Kb deletion 2q13
46XY UPD 2
46XX UPD 6
46XY XP22.32
46XX XP22.33 deletion
46XY XP26.2 duplication
46XX Xq28 deletion
46XX 2p25.3 gain
46XX LOH 41 gene involved
46XX LOH homozygous for mutation in EVC, positive for Ellis Van Creveld
46XYdel17(q23.1-23.3) Deletion 17(q23.1-23.3)
46XX(+3)(p23) 1.8Mb 3pterp26.3, 15.1Mb terminal duplication 15q25.3qter
46XXdel(18)(q11.2) Consistent with karyotype
46XYdel5(p15.1) 19.16Mb deletion (5p15.33p14.13)
46XX del6(q25) Consistent with karyotype
45XYder15t(15;22)(9q10;q10) Consistent with karyotype
46XXdup1(q32q42) Consistent with karyotype
47XY+mar.ish +der(14 or 22)
46,XYinv14 (q13q23.2)
46XY inv(10)(p13q11.2)
47XXY – 3 cases Consistent with karyotype in one case tested
47XYY – 3 cases Consistent with karyotype in one case tested
47XX+marker chromosome
46XX/46XdelXq12 Consistent with karyotype
47XXi(18)(p10)[11]/46XX[12] Consistent with 18p gain
47XX+mar[12]/46XX[37] 12.1Mb add (1p13.3p12)
47XX+13[3]/46XX[17] Consistent with 13 gain
47XX+13 Consistent with 13 gain
47XY+20[10]/46 XY[10]
47XX+21[16]46XX[334] Consistent with 21 gain
47XY+8[2]/46XY[37] Consistent with 8 gain
47XYi7[8]/46XY[12] Consistent with 7p gain
Trisomy 13 – 4 cases Consistent with karyotype in 2 case tested
47XY +16
Trisomy 18 – 32 cases Consistent with karyotype in 9 cases tested
Trisomy 21 – 60 cases Consistent with karyotype in 16 cases tested
46XYder13;14+21 Consistent with 21 gain
45X – 21 cases Consistent with karyotype in 6 cases tested, one case of additional 2q21 deletion.
69XXX or 69XXY – 5 cases

Kb, kilobase; Mb, megabase; UPD, Uni Parental Disomy; LOH, Loss of Heterozygosity.

Shani et al. Chromosomal findings cffDNA could not detect. Am J Obstet Gynecol 2016.


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Division of Reproductive and Medical Genetics, Department of Obstetrics & Gynecology and Women’s Health, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY

Corresponding author: Susan Klugman, MD.

The authors report no conflict of interest.

Cite this article as: Shani H, Goldwaser T, Keating J, et al. Chromosomal abnormalities not currently detected by cell-free fetal DNA: a retrospective analysis at a single center. Am J Obstet Gynecol 2016;214:729.e1-11.