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.
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.
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 ).
| 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 |
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
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
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 ).
| 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% |
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.
