Detection of triploid, molar, and vanishing twin pregnancies by a single-nucleotide polymorphism–based noninvasive prenatal test




Materials and Methods


Patients


The current study included all samples from participating centers received for commercial testing from March 1, through Nov. 30, 2013, that received an NIPT result. This study received a notification of exempt determination from an institutional review board (Ethical and Independent Review Services, no. 14064-01). All samples were analyzed at Natera’s Clinical Laboratory Improvement Act–certified and College of American Pathologists–accredited laboratory in San Carlos, CA. Analysis was performed for all samples on chromosomes 13, 18, 21, X, and Y, and included detection of trisomy 21, trisomy 18, trisomy 13, monosomy X, sex chromosome abnormalities (47,XXX/XXY/XYY), fetal sex, and additional fetal haplotypes.


Sample collection and NIPT


Maternal blood samples (>13 mL) were collected in Streck (Omaha, NE) blood collection tubes and processed at Natera (San Carlos, CA) within 6 days of collection. All samples were accompanied by a requisition form from the ordering clinician, and included the following patient information: gestational age, maternal date of birth, maternal weight, whether it was a multigestation pregnancy, and whether a paternal buccal swab was included. Ordering clinicians determined indication(s) for testing. Cases accepted for analysis were indicated as singleton pregnancies by ordering clinicians. Results were reported directly to the ordering clinician or distribution partners.


Samples were considered outside of the specifications for testing and were not analyzed if there was insufficient blood volume or the wrong tube was used, the sample was damaged, the sample was received at the laboratory >6 days after collection, the gestational age was <9 weeks, the patient used an egg donor, or the patient had a confirmed multiple gestation. Testing was performed on all samples with sufficient blood volume (>13 mL) as described previously using validated laboratory methodologies (cfDNA isolation, polymerase chain reaction amplification targeting 19,488 SNPs, high-throughput sequencing, and analysis using the Next-generation Aneuploidy Test Using SNPs [NATUS] algorithm). Samples were subject to a stringent set of quality-control metrics before reports were sent to ordering clinicians.


The NATUS algorithm incorporates parental genotypic information, uses numerous quality control metrics, and determines a sample-specific accuracy for each interrogated chromosome. Briefly, the algorithm considers parental genotypic information, crossover frequency data, and possible fetal chromosome copy numbers (monosomy/disomy/trisomy) at 19,488 evaluated polymorphic loci. By comparing the observed fetal allele distributions from the sequencing data to the predicted distributions, the algorithm determines the fetal ploidy state with the maximum likelihood for each interrogated chromosome; this maximum likelihood probability is incorporated into a risk score for reporting purposes. The NATUS algorithm is currently only validated to call aneuploidy in singleton gestations. However, the algorithm is able to determine when cfDNA sequencing results do not match the modeled fetal copy numbers with a high likelihood, and can identify the presence of additional fetal haplotypes that indicate either fetal triploidy or the presence of an undetected dizygotic multiple gestation. The presence of an additional fetal haplotype was identified when all tested chromosomes failed to match the disomy hypothesis, and when the additional haplotype was apparent from allele distributions. At this time, the algorithm cannot distinguish dizygotic twin gestations from triploidy pregnancies due to similar allele distributions ( Figure 1 ); therefore these are reported as a single call. Specifically, in a euploid singleton pregnancy, where the maternal alleles are AA (with dimorphic alleles arbitrarily labeled as A and B), the 2 expected fetal genotypes include AA and AB. By contrast, in dizygotic twin and triploid pregnancies where the maternal alleles are AA, there are 3 expected fetal genotypes for both triploid (AAA, AAB, ABB) ( Figure 1 , A) and dizygotic twin (AA/AA, AA/AB, AB/AB) ( Figure 1 , B) pregnancies. This results in equivalent B allele distributions (0, 1, or 2 B alleles), and very similar A allele distributions in triploid (1, 2, or 3) and dizygotic twin (2, 3, or 4) pregnancies.




Figure 1


Additional parental haplotypes detected in vanishing twin and triploidy pregnancies

Graphical representation of sequencing data obtained from A , a paternal triploidy sample and B , a vanishing twin sample. It is important to note that this is not how the algorithm makes copy number calls, but is one method for visualizing data. All interrogated single-nucleotide polymorphisms (SNPs) are assumed to be dimorphic and are designated as A and B for simplicity. Briefly, for each graph, the number of A allele reads as a fraction of total reads is plotted (y-axis) against the position of each of several thousand interrogated SNPs on chromosomes of interest (x-axis). X-axis represents the linear position of each SNP along the chromosome, and each spot corresponds to a single SNP. As plasma cell-free DNA (cfDNA) is a mixture of fetal and maternal cfDNA, the vertical position of each spot represents the sum of contribution of both fetal and maternal allele reads, and is a function of the fetal fraction. To more readily visualize maternal and fetal contributions, spots are colored according to maternal genotype: SNPs for which the mother is homozygous for the A allele (AA) are red , SNPs for which the mother is homozygous for the B allele (BB) are blue , and SNPs for which the mother is heterozygous (AB) are green . Since the majority of plasma cfDNA is maternal in origin, spots mainly distribute according to the maternal genotype. The contribution of fetal allele reads results in segregation into distinct clusters. Because loci targeted on the Y chromosome are homologous to loci on the X chromosome, but differ by 1 nucleotide, probes hybridize to both chromosomes. However, targeted alleles have chromosomally distinct, nondimorphic identities, so are not color-coded; all alleles from the X chromosome are assigned as A alleles, and all alleles from the Y chromosome are assigned as B alleles. A , Confirmed paternal triploidy, 22.6% fetal cfDNA fraction. Position of peripheral red and blue clusters indicates additional haplotypes, as indicated to the right of the plot. Alleles for the X chromosome are indicated (X). Presence of B alleles from the Y chromosome shifts the cluster downward, indicating the presence of a single Y chromosome (based on distribution of reads). Together, this suggests a fetal chromosomal complement of 69,XXY. B , Confirmed vanishing twin, 19.6% fetal cfDNA fraction. Position of peripheral red and blue clusters indicates additional haplotypes, as indicated to the right of the plot. Presence of B alleles from the Y chromosome shifts the cluster downward, indicating the presence of a single Y chromosome (based on distribution of reads). Together, this suggests the presence of vanishing twins, as indicated to right of plot; “Fetus 1/2” indicates genotypes of fetus 1/fetus 2. Center green clusters only segregate and are readily visible at higher fetal fractions.

Curnow. SNP-based NIPT detects vanishing twin pregnancies. Am J Obstet Gynecol 2015 .


For cases with an identified additional fetal haplotype, a report was sent to the ordering clinician or laboratory indicating that the results were consistent with a possible triploid or vanishing twin pregnancy, and recommending follow-up counseling and testing; after report delivery, a Natera genetic counselor contacted the ordering clinician/provider to answer questions related to the NIPT findings.


Clinical outcomes


Follow-up information on cases identified with an additional fetal haplotype was requested by telephone at regular intervals from ordering clinicians and partner laboratories. All information detailing ultrasound findings and pregnancy outcomes were recorded in the laboratory follow-up database. Follow-up information directly reported to Natera by providers was also recorded. Multifetal pregnancies were confirmed by ultrasound, which is consistent with how they are clinically diagnosed in practice. Cases were categorized as follows: (1) “confirmed vanishing twin pregnancy” if ultrasound detected a second empty sac or second sac containing a deceased fetus; (2) “confirmed ongoing twin pregnancy” if ultrasound showed an ongoing and viable twin pregnancy; (3) “confirmed fetal triploidy” if triploidy was confirmed by invasive testing or testing of products of conception (POC); (4) “unconfirmed fetal triploidy” included cases without invasive diagnostic testing but with ultrasound findings consistent with triploidy; (5) “confirmed nontriploid pregnancy” included cases where invasive diagnostic testing ruled out fetal triploidy and there was no evidence of co-twin demise; (6) “pregnancy loss” for cases where patients experienced spontaneous abortion and did not obtain karyotype confirmation; or (7) “no follow-up” where follow-up information was requested but was not received by the time of manuscript submission.


Statistical analysis


Differences in the maternal age and gestational age between confirmed twin and confirmed vanishing twin cohorts were determined using a Mann-Whitney rank sum test. A t test was used to compare the fetal fraction in confirmed twin and vanishing twin cases. SigmaPlot 12.5 (Systat Software, San Jose, CA) was used for all statistical analyses. A P value of < .05 was considered statistically significant. Unless otherwise indicated, data are presented as the mean ± SD.




Results


Study participants and samples


In the present cohort of 30,795 cases with an NIPT result, 130 (0.42%) received a report indicating the presence of additional fetal haplotypes. For the whole cohort, the mean maternal age was 33.6 ± 6.1 (range, 13.0–63.0) years ( Figure 2 , A), and the mean gestational age was 14.5 ± 4.7 (range, 9.0–40.9) weeks ( Figure 2 , B); maternal age was confirmed for the single case with a maternal age >52 years. For the 130 cases where an additional fetal haplotype was identified by NATUS, the mean maternal age was 34.3 ± 5.7 (range, 19.0–52.0) years ( Figure 2 , C), and the mean gestational age was 13.3 ± 4.1 (range, 9.0–38.0) weeks ( Figure 2 , D). While the majority of NIPT samples were from women at early gestational ages, samples were received up to 40 weeks’ gestation ( Figure 3 ); 2% (658/30,795) of samples were from women in their third trimester.




Figure 2


Maternal age and gestational age histograms

A , Maternal and B , gestational age histograms for the whole cohort. C , Maternal and D , gestational age histograms for cases with identified additional fetal haplotypes. Although not apparent because of the scale of the Y-axis, within the whole cohort there were 8 cases with maternal ages of 49-63 years; maternal age was confirmed for the single case with a maternal age of >52 years.

Curnow. SNP-based NIPT detects vanishing twin pregnancies. Am J Obstet Gynecol 2015 .



Figure 3


Graphical representation of time elapsed between estimated fetal demise and detection by NIPT for 5 confirmed vanishing twin cases

Each line indicates an individual confirmed vanishing twin case with a known estimated date of fetal demise. The longest period between fetal demise and NIPT was 8 weeks.

NIPT , noninvasive prenatal testing.

Curnow. SNP-based NIPT detects vanishing twin pregnancies. Am J Obstet Gynecol 2015 .


Clinical outcomes


Karyotype or ultrasound confirmation (karyotype for singleton pregnancies, ultrasound for multifetal pregnancies) was available for 76 (58.5%) of the 130 cases identified with additional parental haplotypes. This included 32 (42.1%) vanishing twin, 37 (48.7%) viable twin, 4 (5.3%) triploid pregnancies, and 3 (3.9%) nontriploid pregnancies that lacked evidence of co-twin demise ( Table 1 ). For the 3 nontriploid pregnancies, 2 had euploid karyotypes, and 1 was shown to be a trisomy 18 fetus ( Appendix ; Supplementary Table ).



Table 1

Follow-up information on twins/triploidy calls





































All cases (n = 130 a ) Value
Multifetal pregnancies
Ongoing twin b 37
Vanishing twin b 32
Singleton pregnancies
Confirmed triploid c 4
Unconfirmed triploid 1 d
Confirmed nontriploid 3 e
Unknown fetal no.
Pregnancy loss f 3
No follow-up g 50

Curnow. SNP-based NIPT detects vanishing twin pregnancies. Am J Obstet Gynecol 2015 .

a Total no. of cases with “additional fetal haplotypes” (twins/triploidy) result at ≥9 wk of gestation


b Confirmed by ultrasound detection of multifetal pregnancy


c Confirmed by karyotype


d Patient had ultrasound findings that were consistent with triploid fetus, amniocentesis was not possible because of oligohydramnios


e Invasive testing revealed single euploid fetus in 2 pregnancies and single trisomy 18 fetus in 1 pregnancy


f Patient experienced spontaneous abortion and did not obtain karyotype confirmation, ultrasound was suggestive of singleton pregnancy


g Follow-up information was not available.



Multifetal pregnancies


Vanishing twin cases had a significantly higher median maternal age than twin cases, 37.5 and 33.0 years, respectively ( P < .001). The median gestational age was slightly lower in vanishing twin cases than in twin cases, 12.1 and 13.0 weeks, respectively ( P = .018). There was no significant difference ( P = .686) between the average fetal fraction of vanished twin (11.0 ± 3.8%) and twin (11.4 ± 4.3%) pregnancies. Of the 32 vanishing twin cases, 25 (78.1%) were in the first trimester and 7 (21.9%) were in the second trimester at the time of NIPT sampling. Five cases reported an estimated date of fetal demise: demise occurred in the first trimester in all 5 cases ( Figure 3 ). The time between demise and NIPT sampling ranged from 2-8 weeks ( Table 2 ).



Table 2

Case details for confirmed vanishing twin cases








































Case GA at demise, wk GA at NIPT, wk Time from demise to NIPT, wk Fetal fraction
1 8.0 10.3 2.3 11.7%
2 7.1 10.4 3.3 4.6%
3 8.6 12.6 4.0 12.8%
4 8.0 14.7 6.7 11.8%
5 7.0 15.0 8.0 8.1%

GA at estimated date of co-twin demise and at time of sample collection for NIPT, elapsed time between estimated demise and NIPT sampling, and fetal fraction for confirmed vanishing twin cases where clinical follow-up provided estimated date of fetal demise.

GA , gestational age; NIPT , noninvasive prenatal testing.

Curnow. SNP-based NIPT detects vanishing twin pregnancies. Am J Obstet Gynecol 2015 .


Triploid pregnancies


All triploidy cases in this cohort were determined to be diandric ( Table 3 ), indicating that in each case the additional fetal haplotype was paternal in origin. Fetal sex was determined for all triploidy cases by analysis of fetal sex chromosome copy numbers; the fetal karyotype matched the fetal sex determined by NIPT for all 3 triploidy cases where karyotype specifics were communicated during follow-up ( Table 3 ). For triploidy cases 1, 2, and 4 detailed in Table 3 , the pregnancies spontaneously aborted and karyotype confirmation was obtained from the POC; during clinical follow-up, 2 of these cases were reported as partial mole pregnancies. For triploidy cases 3 and 5 ( Table 3 ), clinical evaluation identified large placentas and oligohydramnios in both cases.


May 10, 2017 | Posted by in GYNECOLOGY | Comments Off on Detection of triploid, molar, and vanishing twin pregnancies by a single-nucleotide polymorphism–based noninvasive prenatal test
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