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First-trimester screening in triplets

Published:August 29, 2011DOI:https://doi.org/10.1016/j.ajog.2011.06.107

      Objective

      The purpose of this study was to determine the performance of Down syndrome screening in triplet pregnancy.

      Study Design

      Nuchal translucency (NT; n = 794), nasal bone (n = 219), and biochemistry (n = 198) were evaluated in triplet pregnancy. Screening performance was evaluated with the use of delta and Gaussian models.

      Results

      The median multiples of the median values for free beta human chorionic gonadotropin and pregnancy-associated plasma protein A were 2.86 and 3.48, respectively. A significant correlation in delta NT within pregnancy was observed (0.46-0.68). The modeled false-positive rates were 11.7%, 7.4%, and 8.9% with the delta model and 11.9%, 6.6%, and 12.0% with the Gaussian model for NT, NT + nasal bone, and NT + biochemistry. Based on simulation, the detection rate at 12 weeks' gestation was 78%, 93%, and 80% for NT, NT + nasal bone, and NT + biochemistry at a 10% false-positive rate using either the delta or Gaussian models.

      Conclusion

      In triplet pregnancy, the addition of nasal bone lowers the false-positive rate of nuchal translucency screening. More data are required on the effectiveness of biochemistry.

      Key words

      The incidence of triplet and higher order multifetal gestation in the United States has risen several hundred percent since 1980, primarily because of the increasingly widespread availability of fertility therapies.
      • Evans M.I.
      • Britt D.W.
      Selective reduction in multifetal pregnancies.
      • Martin J.A.
      • Hamilton B.E.
      • Sutton P.D.
      • et al.
      Births: final data for 2006 Natl Vital Stat Rep 2009.
      The natural incidence of spontaneous triplet pregnancy is approximately 1 in 8000 births
      • Guttmacher A.F.
      The incidence of multiple births in man and some of the other unipara.
      ; however, approximately 95% of triplet pregnancies now come from infertility therapies. By comparison, triplet births accounted for 1 in 2000 pregnancies in 2006.
      • Evans M.I.
      • Britt D.W.
      Selective reduction in multifetal pregnancies.
      • Martin J.A.
      • Hamilton B.E.
      • Sutton P.D.
      • et al.
      Births: final data for 2006 Natl Vital Stat Rep 2009.
      Because women who carry triplet pregnancies also tend to be older than those who carry singleton pregnancies, the questions of prenatal screening and diagnostic procedures have become more prominent. Most maternal serum markers that are used in prenatal screening are associated with the number of fetuses in utero with the median multiples of the median (MoM) values approximately equal to the number of fetuses, although some significant variation from those levels is well known.
      • O'Brien J.E.
      • Dvorin E.
      • Yaron Y.
      • et al.
      Differential increases in AFP, hCG, and uE3 in twin pregnancies: impact on attempts to quantify Down syndrome screening calculations.
      • Spencer K.
      • Nicolaides K.H.
      Screening for trisomy 21 in twins using first trimester ultrasound and maternal serum biochemistry in a one-stop clinic: a review of three years experience.
      As a consequence, screening beyond twin pregnancies with second-trimester maternal serum has never been practical.
      For Editors' Commentary, see Table of Contents
      First-trimester screening for chromosome abnormalities with the use of free beta human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein A (PAPP-A), and nuchal translucency (NT) now is offered routinely to patients in the United States, and first-trimester screening is replacing second-trimester screening as the primary method of evaluation.
      • Fang Y.M.
      • Benn P.
      • Campbell W.
      • Bolnick J.
      • Prabulos A.M.
      • Egan J.F.
      Down syndrome screening in the United States in 2001 and 2007: a survey of maternal-fetal medicine specialists.
      In addition, some centers can now provide an assessment of nasal bone during the ultrasound examination.
      • Cicero S.
      • Curcio P.
      • Papageorghiou A.
      • Sonek J.
      • Nicolaides K.
      Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study.
      The combination of biochemical and ultrasound screening initially was introduced primarily for singleton pregnancies. In multifetal pregnancies, the evaluation of ultrasound markers has advantages because an assessment of each fetus can be determined. However, the biochemical assessment applies to the pregnancy as a whole and cannot be applied directly to the individual fetus because there is no way to differentiate among the contributions of each fetus to the total. A number of reports have demonstrated that the addition of biochemical testing to NT assessment in twin pregnancy leads to improved screening performance, although in dizygotic twins the screening performance is still not as good as in singleton pregnancy.
      • Spencer K.
      Screening for trisomy 21 in twin pregnancies in the first trimester using free beta-hCG and PAPP-A, combined with fetal nuchal translucency thickness.
      • Cleary-Goldman J.
      • Rebarber A.
      • Krantz D.
      • Hallahan T.
      • Saltzman D.
      First-trimester screening with nasal bone in twins.
      • Goncé A.
      • Borrell A.
      • Fortuny A.
      • et al.
      First-trimester screening for trisomy 21 in twin pregnancy: does the addition of biochemistry make an improvement?.
      The diminished screening performance in dizygotic twin pregnancy is because the biochemical markers will blend the contribution of the 2 fetuses. For example, for a marker that is 1 MoM in a normal singleton pregnancy and 2 MoM in an affected singleton pregnancy, the combination in a normal twin should model approximately 2 (1 + 1); in a twin pregnancy with 1 affected fetus in whom the analyte is elevated in Down syndrome pregnancies, the combination becomes 3 (1 + 2). Dividing the total by 2, the affected MoM is now 1.5 times that of the unaffected and therefore not as elevated as is observed in singleton (2.0) pregnancy. Similarly if the marker is 0.5 in a Down syndrome–affected singleton pregnancy, then the expected difference in twins discordant for Down syndrome would be just 0.75. Regardless, modeling and real patient data show that the addition of biochemical screening in twin pregnancies lowers the false-positive rate compared with ultrasound screening alone.
      • Cuckle H.S.
      • Arbuzova S.
      Multimarker maternal serum screening for chromosomal abnormalities Genetic disorders and the fetus.
      • Spencer K.
      • Bindra R.
      • Nix A.B.
      • Heath V.
      • Nicolaides K.H.
      Delta-NT or NT MoM: which is the most appropriate method for calculating accurate patient-specific risks for trisomy 21 in the first trimester?.
      The difficulties that are involved with the use of biochemistry in twin pregnancies are further exacerbated in higher-order multiple pregnancies. With similar logic as described for twins, in triplet pregnancies the elevation that is derived from a single affected fetus is likely to be masked even further so that the elevation is only 1.33 (4/3) times that of the unaffected. However, with a large enough database on triplet pregnancies, this small differential may be enough to benefit the addition of biochemical screening to ultrasound screening. Recently, many centers have begun to evaluate triplet pregnancies using ultrasound markers alone. Here, we analyze a large set of triplet pregnancies for NT and nasal bone and evaluate the potential addition of biochemical screening.

      Materials and Methods

      We performed a retrospective analysis of all 794 sets of triplets between January 2003 and July 2010 in which NT measurement was performed and provided to the laboratory. Among these triplet pregnancies, nasal bone evaluation was performed in 219 patients, and dried blood was collected from 198 patients. NT was measured in all 3 fetuses for all cases, and nasal bone was measured in all 3 fetuses for 202 of the 219 pregnancies (92%) in which nasal bone was evaluated. All sonographers participated in either the Fetal Medicine Foundation or Nuchal Translucency Quality Review programs. For the 198 patients with dried blood specimens, 145 specimens were analyzed with in-house enzyme-linked immunosorbent assays for free β-hCG and PAPP-A, and 53 specimens were analyzed with in-house dual analyte DELFIA assays (Perkin Elmer Life Sciences, Boston, MA). The Institutional Review Board of Mount Sinai determined that the study did not constitute human research under Department of Health and Human Services and Food and Drug Administration regulations and was thus exempt.
      The patients in the study had an average gestational age of 87.85 days (SD, ± 4.18 days) and an average maternal age of 33.5 ± 4.8 years. The subgroup that had nasal bone evaluation performed had an average gestational age of 86.45 ± 4.23 days and an average maternal age of 33.95 ± 5.0 years. The subgroup that had biochemistry performed had an average gestational age of 87.0 ± 4.36 days and an average maternal age of 33.48 ± 4.9 years.
      Down syndrome risks were calculated for NT, NT + nasal bone, and NT + biochemistry. Only 11 patients had NT, nasal bone, and biochemistry so there was not enough power to evaluate the combination. Risks were calculated with the delta model. Namely, NT likelihood ratios were determined on the basis of delta values with the use of the algorithm for singleton pregnancies,
      • Spencer K.
      • Bindra R.
      • Nix A.B.
      • Heath V.
      • Nicolaides K.H.
      Delta-NT or NT MoM: which is the most appropriate method for calculating accurate patient-specific risks for trisomy 21 in the first trimester?.
      and this likelihood ratio was applied to each fetus. Nasal bone likelihood ratios were determined with a published formula
      • Cicero S.
      • Rembouskos G.
      • Vandecruys H.
      • Hogg M.
      • Nicolaides K.H.
      Likelihood ratio for trisomy 21 in fetuses with absent nasal bone at the 11-14-week scan.
      ; if any of the 3 fetuses did not have nasal bone assessment, then a likelihood ratio of 1 was used for that fetus. Biochemistry was factored in to the risk algorithm with the approach that Spencer and Nicolaides
      • Spencer K.
      • Nicolaides K.H.
      First trimester prenatal diagnosis of trisomy 21 in discordant twins using fetal nuchal translucency thickness and maternal serum free beta-hCG and PAPP-A.
      reported for twin pregnancies. The free β-hCG and PAPP-A MoM values were divided by the median MoM value that was observed in triplet pregnancies. The adjusted MoM values were input into the singleton risk algorithm for biochemistry
      • Orlandi F.
      • Rossi C.
      • Orlandi E.
      • et al.
      First-trimester screening for trisomy-21 using a simplified method to assess the presence or absence of the fetal nasal bone.
      to determine the biochemical likelihood ratio. For the combined NT and nasal bone protocol, the likelihood ratio for NT and nasal bone in each fetus were multiplied together to determine the overall likelihood ratio. For the combined NT and biochemistry protocol, the NT likelihood ratio for each fetus was multiplied by the biochemistry likelihood ratio to determine the overall likelihood ratio for each fetus. The overall likelihood ratio was multiplied by the previous risk of Down syndrome to get the posterior Down syndrome risk.
      Risk for triplet pregnancies was also evaluated with a Gaussian model. The parameters for the NT distribution were based on previously published parameters.
      • Cuckle H.
      • Benn P.
      • Wright D.
      Down syndrome screening in the first and/or second trimester: model predicted performance using meta-analysis parameters.
      The parameters for the biochemical distribution were the same as in singleton pregnancies, except for the means. In the unaffected distribution, the mean log (MoM) of each analyte was set equal to the log of the median MoM that was observed in triplet pregnancies. For the affected distribution, an estimate of the median for each analyte was determined by the estimation of the median to be the sum of 2 plus the affected median in singleton pregnancy and then normalization of this value by multiplication by the ratio of the unaffected median in triplet pregnancies to 3.0. The affected mean was then set equal to the log of the estimated median.
      For comparative purposes, 275,690 singleton pregnancies and 7455 dichorionic twin pregnancies in which both dried blood biochemistry using the DELPHIA assay and NT were performed between November 2009 and September 2010 were evaluated.
      A risk cutoff of 1 in 300 pregnancies was used; a pregnancy was considered to be at risk if the risks for any of the fetuses in the pregnancy were above the cutoff point. Age-adjusted false-positive rates were modeled with the observed likelihood ratios and the age distribution of live births for singleton, twin, and triplet pregnancies as appropriate. Age-adjusted false-positive rates for triplet pregnancies were also modeled by application of the age distribution for singleton pregnancies to correct for the maternal age effect because triplet pregnancies were older than singleton pregnancies. In modeling for twin and triplet pregnancies, 1 year was subtracted from the age to account for use of egg donors. The 1-year adjustment factor was based on a review of our most recent 6-month data on multiple pregnancies in which the average age (based on the patient's date of birth) was 1 year greater than the average age based on the patient's eggs.
      Detection rates for 1 affected triplet were estimated for testing at 12 weeks' gestation by simulation of 100,000 sets of MOM values from the affected distributions that were described earlier and then application of the likehood ratio calculations described earlier. The simulated NT MoM values were adjusted by multiplication by the observed unaffected median.

      Results

      The median MoM values for free β-hCG and PAPP-A were 2.86 (95% confidence interval [CI], 2.69–3.08) and 3.48 (95% CI, 3.24–3.91), respectively. The median delta NT values for triplet pregnancies A, B, and C were −.12 (95% CI, −0.14 to −0.07), −.09 (95% CI, −0.11 to −0.06), and −0.09 (95% CI, −0.11 to −0.06) respectively. The median delta NT of all measurements was −0.10 (95% CI, −0.12 to −0.08). The Spearman rank correlation coefficient between the minimum and median, the minimum and maximum, and the median and maximum delta NT in each triplet pregnancy was 0.65, 0.46, and 0.68, respectively. With the use of MoMs, the median NT MoM was 0.93 (95% CI, 0.91–0.95), 0.94 (95% CI, 0.93–0.96), 0.93 (95% CI, 0.91–0.95) in triplets A, B, and C, respectively. The overall median NT MoM was 0.93 (95% CI, 0.92–0.95). The Spearman rank correlation coefficient between the minimum and median, the minimum and maximum, and the median and maximum NT MoM in each triplet pregnancy was 0.8476, 0.6704, and 0.7918, respectively. In all cases in which nasal bone was assessed, the nasal bone was present.
      Screening with NT alone, 103 of 794 triplet pregnancies (13.0%) were at risk for Down syndrome, compared with 5.8% and 9.0% in singleton and twin pregnancies, respectively (Table 1). Among the triplet pregnancies at increased risk, 63 pregnancies had 1 fetus at risk; 19 pregnancies had 2 fetuses at risk, and 21 pregnancies had 3 fetuses at risk. Overall, 164 of 2382 triplet fetuses (6.9%) were at risk for Down syndrome. When nasal bone was added to NT, the screen positive rate dropped to 11.9% of pregnancies (26/219; Table 2), compared with 2.8% and 4.7% in singleton and twin pregnancies, respectively. The addition of the biochemical markers to NT resulted in a 12.1% false-positive rate (Table 3), compared with 5.3% and 6.4% in singleton and twin pregnancies, respectively.
      TABLE 1Pregnancies and fetuses in triplet pregnancy with risk of >1 in 300 pregnancies, based on nuchal translucency
      VariableSingletonTwinTriplet
      Delta modelGaussian model
      Pregnancies/fetusesPregnanciesFetusesPregnanciesFetusesPregnanciesFetuses
      Total, n275,690745514,91079423827942382
      Fetuses at risk, n (%)
       115,878 (5.8)441 (5.9)441 (3.0)63 (7.9)63 (2.6)74 (9.3)74 (3.1)
       2N/A228 (3.1)456 (3.1)19 (2.4)38 (1.6)26 (3.3)52 (2.2)
       3N/AN/AN/A21 (2.8)63 (2.8)17 (2.1)51 (2.1)
       Total, n (%)15,878 (5.8)669 (9.0)897 (6.0)103 (13.0)164 (6.9)117 (14.7)177 (7.4)
      Age-adjusted, %
      Rates determined by modeling the observed likelihood ratios with the age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      3.77.24.911.76.511.96.4
      N/A, not applicable.
      Krantz. First-trimester triplets. Am J Obstet Gynecol 2011.
      a Rates determined by modeling the observed likelihood ratios with the age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      TABLE 2Pregnancies and fetuses in triplet pregnancy with risk of >1 in 300, based on nuchal translucency + nasal bone
      VariableSingletonTwinTriplet
      Delta modelGaussian model
      Pregnancies/fetusesPregnanciesFetusesPregnanciesFetusesPregnanciesFetuses
      Total, n121,27731876374219657219657
      Fetuses at risk, n (%)
       13414 (2.8)124 (3.9)124 (1.9)17 (7.8)17 (2.6)15 (6.8)15 (2.3)
       2N/A0.8 (26)52 (0.8)3 (1.4)6 (0.9)3 (1.4)9 (1.4)
       3N/AN/AN/A6 (2.7)18 (2.7)5 (2.3)15 (2.3)
       Total, n (%)3414 (2.8)150 (4.7)176 (2.8)26 (11.9)41 (6.2)23 (10.5)39 (5.9)
      Age-adjusted, %
      Rates determined by modeling the observed likelihood ratios with the Age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      2.04.12.77.43.76.63.3
      N/A, not applicable.
      Krantz. First-trimester triplets. Am J Obstet Gynecol 2011.
      a Rates determined by modeling the observed likelihood ratios with the Age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      TABLE 3Pregnancies and fetuses in triplet pregnancy with risk of >1 in 300, based on nuchal translucency + biochemistry
      VariableSingletonTwinTriplet
      Delta modelGaussian model
      Pregnancies/fetusesPregnanciesFetusesPregnanciesFetusesPregnanciesFetuses
      Total, n275,690745514,910198594198594
      Fetuses at risk, n (%)
       14605 (5.3)254 (3.4)254 (1.7)12 (6.1)11 (1.9)25 (12.6)25 (4.2)
       2N/A219 (2.9)2.9 (438)6 (3.0)12 (2.0)4 (2.0)8 (1.3)
       3N/AN/AN/A6 (3.0)21 (3.5)4 (2.0)12 (2.0)
       Total, n (%)4605 (5.3)473 (6.4)692 (4.6)24 (12.1)44 (7.4)33 (16.7)45 (7.6)
      Age-adjusted, %
      Rates determined by modeling the observed likelihood ratios with the Age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      3.74.83.58.95.212.06.4
      N/A, not applicable.
      Krantz. First-trimester triplets. Am J Obstet Gynecol 2011.
      a Rates determined by modeling the observed likelihood ratios with the Age distribution of live births for the respective cohorts; for twin and triplet pregnancies, the age distribution was shifted by 1 year to account for egg donors.
      Modeling the data for the US maternal age distribution in triplet pregnancies resulted in false-positive rates of 11.7%, 7.4%, and 8.9% with the delta method and 11.9%, 6.6%, and 12% with the Gaussian method for NT only, NT + nasal bone, and NT + biochemistry, respectively. These rates were significantly higher than the 3.7%, 2.0%, and 3.7% that were observed in singleton pregnancy, respectively, and the 7.2%, 4.1% and 4.8% that were observed in twin pregnancy, respectively.
      A significant proportion of the false-positive rate is secondary to the fact that triplet pregnancies tend to be older than singleton pregnancies. Correction for the maternal age difference by modeling the triplet data with the age distribution of singleton pregnancies showed that the false-positive rate decreased by 2.3%, 1.8%, and 2.3% for NT only, NT + nasal bone, and NT + biochemistry, respectively, when the age distribution for singleton pregnancy was used instead of the age distribution of triplet pregnancy.
      Using simulation, an estimate of detection efficiency at a fixed 10% false-positive rate was estimated to be 78%, 93%, and 80% with the use of NT, NT + nasal bone and NT + biochemistry in either the delta model or the Gaussian model.

      Comment

      Screening for Down syndrome with the use of ultrasound markers in multiple pregnancies has long been established.
      • Cicero S.
      • Curcio P.
      • Papageorghiou A.
      • Sonek J.
      • Nicolaides K.
      Absence of nasal bone in fetuses with trisomy 21 at 11-14 weeks of gestation: an observational study.
      The advantage of ultrasound screening in multiple pregnancies is that it allows for risks to be calculated on a per-fetus basis. However, the risk on a per-pregnancy basis (the risk of at least 1 affected fetus) increases with the number of fetuses. Our data show that the addition of nasal bone to NT improves screening performance. All of the nasal bone evaluations in this study indicated that nasal bone was present. It is possible that, in situations in which the nasal bone was absent, the laboratory was not asked to provide a risk assessment; thus, there is the potential that the false-positive rate with nasal bone in actual practice might be higher than in the present study.
      In twin pregnancies, the addition of maternal serum free β-hCG and PAPP-A to the ultrasound markers can lower the false-positive rate on a per-pregnancy basis
      • Cleary-Goldman J.
      • Rebarber A.
      • Krantz D.
      • Hallahan T.
      • Saltzman D.
      First-trimester screening with nasal bone in twins.
      • Goncé A.
      • Borrell A.
      • Fortuny A.
      • et al.
      First-trimester screening for trisomy 21 in twin pregnancy: does the addition of biochemistry make an improvement?.
      relative to NT alone. Our data now indicate that the addition of biochemical markers may also be beneficial in triplet pregnancy. Using the delta model, the false-positive rate was reduced by adding biochemistry, with only a small loss in detection rate. With the Gaussian model, false-positive rates were similar, but the detection rate was estimated to be higher. As a result, by improving the risk calculation model or adjusting cut-offs, it may now be feasible to include biochemistry in the risk assessment model for triplet pregnancies. In addition, in pregnancies in which 2 of 3 or all 3 fetuses are affected, the performance of biochemistry would be expected to be better than in a pregnancy in which only 1 fetus is affected, although such pregnancies would be rare. The combination of biochemistry with both NT and nasal bone is still to be evaluated.
      Part of the increase in screen positives that are observed in triplet pregnancies on a per-pregnancy basis is due to the fact that women who carry triplet pregnancies tend to be older than the general pregnant population. Another more significant reason is that any of the 3 fetuses might be at increased risk. If the 3 NT values were independent, the rate would be expected to nearly triple; however, because there is correlation between the NT values and the risk result for each fetus who shares the same maternal age, nasal bone (at least in this study), and biochemistry values, the observed increase in the pregnancy screen positive rate is actually approximately 2-fold relative to the screen positive rate in singleton fetuses. This correlation also counterintuitively caused relatively more pregnancies to have 3 at-risk fetuses than 2 at-risk fetuses.
      Data in twin pregnancies indicate that biochemistry levels in monochorionic pregnancy are different than those in dichorionic pregnancy. In addition, twin pregnancies that experience twin-to-twin transfusion syndrome have an increased chance of having 1 fetus with elevated NT. We did not have data available on chorionicity in the cases that were studied to evaluate these issues in triplet pregnancy, so they must be evaluated in future studies.
      Our data on biochemistry are not clear enough to suggest that it may add to the screening efficacy in triplet pregnancies. However, in natural occurring triplet pregnancy
      • Guilherme R.
      • Drunat S.
      • Delezoide A.L.
      • Oury J.F.
      • Luton D.
      Zygosity and chorionicity in triplet pregnancies: new data.
      in which the incidence of monochorionic twin pairs is more common and there is the possibility of 2 or 3 affected fetuses in the pregnancy, biochemistry may play a role because there would be greater discrimination between these pregnancies and unaffected pregnancies than are seen with 1 affected fetus. Ultimately, patients with multiple pregnancy have to decide whether to rely on a diminished screening performance or having diagnostic procedures that only a small number of clinicians can perform well to try to assess the health of their offspring.

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