While prenatal cfDNA screening has been available for well over a decade, the data surrounding screening in triplet pregnancies is still limited compared to the data available for singleton pregnancies and even twins. However, this is juxtaposed with a true lack of aneuploidy screening options (beyond ultrasound) in triplets. Triplet pregnancies are already accompanied by several unique clinical considerations and challenges, and the risks for significant pregnancy complications are higher. As providers, how can genetic counselors and other health care professionals navigate aneuploidy screening for these cases?

What Professional Guidelines Say

In a 2021 position statement from the International Society for Prenatal Diagnosis (ISPD) on cfDNA screening in multifetal pregnancies, the authors estimate that to have 10 triplet cases with trisomy 21, approximately 11 million total pregnancies would need screening via prenatal cfDNA, which illustrates the mathematical challenge in understanding test performance.¹ This raises the question: will we ever truly have enough data? Or can we rely on what we know and understand about biology as it relates to cfDNA technology and extrapolate from the data in singletons and twins? The authors of the ISPD position statement suggest just that. Most other professional societies do not specifically address cfDNA screening in triplet pregnancies, despite the limited alternatives. 

What Recent Studies Suggest

Since that position statement, a few studies with data on triplet pregnancies have been published. Zakaria et al. analyzed a cohort of 255 triplet pregnancies with cfDNA screening; neonatal outcomes were available in 165 (64.7%).² This cohort included three cases with cfDNA screening results suggestive of trisomy 21, with two cases confirmed (one via prenatal diagnosis and one confirmed at birth) and the third determined to be a false positive. A case of trisomy 18 was also confirmed in the cohort; no cases of trisomy 13 were present. There were no false negatives.²

Soster et al. looked at a cohort of over 1,500 cfDNA samples from triplet pregnancies, with diagnostic or pregnancy outcomes available in 147 (9.43%).³ Out of 13 screen-positive cases, 11 were confirmed by diagnostic testing (10 trisomy 21 cases and one trisomy 18 case). The two remaining cases did not have enough information available to determine concordance but had suggestive ultrasound findings in one case (fetal growth restriction in a case with cfDNA results suggesting a chromosome 18 copy number variation), and a reported fetal demise in the other case (high risk for trisomy 21, one fetal demise and two normal newborns). No false negatives were reported.³

Lastly, a study by van Eekhout et al. looked at 75 cases with triplet pregnancies as part of a larger multifetal cohort.⁴ One case had a cfDNA result suggestive of trisomy 21, which was confirmed. Again, no false negatives were reported.⁴

No-call Rate and Counseling Considerations

The failure rate in the above studies ranges from 2.4%-17.3%.^2-4 Given that many labs may have more stringent reporting criteria for multifetal pregnancies, a higher no-call rate is not surprising. As such, patients should be counseled about no-call rates, ideally with laboratory or platform-specific data for their pregnancy type (in this case, triplets).  

Looking Forward and Ongoing Data Collection

Although the best scenario would be to have even more data surrounding cfDNA performance in triplet pregnancies, the recent literature suggests that cfDNA screening (in combination with routine ultrasounds) in triplet pregnancies can be a reasonable approach, particularly with appropriate patient counseling regarding the benefits and limitations compared to ultrasound alone. We should also acknowledge that this is a patient cohort with less available data, and as such, we encourage providers and laboratories to continue to collect and report on these outcomes.

Clinical Application Through Labcorp’s Solution for Multifetal Pregnancies

Labcorp’s MaterniT® 21 PLUS enables cfDNA screening for all pregnant patients, not just for singletons and twins, but also for triplet and higher-order multiple gestations. It is the only U.S.-based prenatal cfDNA screening test with this capability. To learn more, visit our resources page.

References

1. Palomaki, G. E., Chiu, R. W. K., Pertile, M. D., Sistermans, E. A., Yaron, Y., Vermeesch, J. R., Vora, N. L., Best, R. G., & Wilkins-Haug, L. (2021). International Society for Prenatal Diagnosis position statement: Cell-free (cf) DNA screening for Down syndrome in multiple pregnancies. Prenatal Diagnosis, 41(10), 1222–1232. https://doi.org/10.1002/pd.5832

2. Zakaria, H., Kleinfinger, P., Lohmann, L., Costa, J. M., Tsatsaris, V., Salomon, L. J., Jouannic, J. M., Rosenblatt, J., Demain, A., Benachi, A., El Khattabi, L., & Vivanti, A. J. (2024). Performance of cell-free DNA testing for common fetal trisomies in triplet pregnancies. Prenatal Diagnosis, 44(5), 555–561. https://doi.org/10.1002/pd.6548

3. Soster, E., Dyr, B., Caldwell, S., Moore, S., Almasri, E., Magharyous, H., Saldivar, J. S., & Cacheris, P. (2025). Clinical laboratory experience with prenatal cfDNA screening in triplet pregnancies. Prenatal Diagnosis, 45(6), 705–712. https://doi.org/10.1002/pd.6812

4. van Eekhout, J. C. A., Bax, C. J., Schuurman, L. V. P., Becking, E. C., van der Ven, A. J. E. M., Van Opstal, D., Boon, E. M. J., Macville, M. V. E., Bekker, M. N., & Galjaard, R. J. H. (2025). Performance of non-invasive prenatal testing in vanishing-twin and multiple pregnancies: Results of TRIDENT-2 study. Ultrasound in Obstetrics & Gynecology. https://doi.org/10.1002/uog.70015

Erica L. Soster, MS, LCGC

Soster is a board-certified and licensed genetic counselor living in Indianapolis, Indiana. She completed her B.S. in human biology at Indiana University before receiving her M.S.in medical and molecular genetics at Indiana University. Before working for Labcorp, she worked as a prenatal genetic counselor at the Center for Prenatal Diagnosis at St. Vincent Women’s Hospital in Indianapolis. Soster has a passion for research and data analysis. During her more than nine years with Labcorp, she has authored multiple publications and abstracts related to cfDNA, with particular interest in genome-wide cfDNA, mosaicism and rare aneuploidies.