Non-Invasive Prenatal Testing (NIPT) is a screening tool that analyzes cell-free DNA (cfDNA) circulating in the mother’s bloodstream, providing early insights into the fetus’s genetic makeup. While NIPT is utilized for screening chromosomal abnormalities, it also determines fetal sex early in pregnancy. Determining sex in a twin pregnancy introduces unique technical complexities compared to a singleton pregnancy, stemming from the presence of two distinct fetal DNA contributions.
How NIPT Determines Fetal Sex
NIPT determines fetal sex by detecting the presence or absence of the Y chromosome. The blood sample contains a mixture of the pregnant person’s DNA and cell-free fetal DNA (cffDNA) originating from the placenta. Laboratory analysis quantifies chromosomal sequences within this mixed sample.
If Y chromosome sequences are detected, it confirms at least one fetus is male. If only X chromosomes are detected above background levels, the result indicates both fetuses are female. While this method is highly accurate in singleton pregnancies (over 99%), the presence of two sources of cffDNA significantly complicates the interpretation of the Y chromosome signal in twins.
The Unique Complexity of Twin DNA Analysis
Twin pregnancies challenge NIPT because the maternal blood sample contains a mixed DNA profile from the mother and two separate fetuses. The primary technical limitation involves the Fetal Fraction (FF), which is the proportion of total cell-free DNA belonging to the fetuses. NIPT requires the FF to be above a threshold, often 4%, for reliable results.
In twins, the total FF is the sum of contributions from both fetuses and is generally higher than in singletons. However, the individual contribution of each twin can vary widely, and standard NIPT methods struggle to determine the exact proportion from each. If one twin contributes a disproportionately low amount of cffDNA, their genetic information may be obscured by the other twin’s DNA, potentially leading to an inaccurate result. This disparity in individual FF makes NIPT sex determination more complex, especially in dizygotic (fraternal) twins.
Accuracy Based on Twin Type
The accuracy of NIPT for sex determination depends heavily on the type of twin pregnancy, or zygosity. Twins are classified as either identical (monozygotic) or fraternal (dizygotic), which determines how the NIPT result must be interpreted.
Identical (Monozygotic) Twins
Identical twins develop from a single fertilized egg and share the exact same genetic material, including sex chromosomes. If one twin is male, the other must be male; if one is female, the other must be female. The NIPT test only needs to confirm the presence or absence of the Y chromosome, similar to a singleton pregnancy. Consequently, the accuracy of NIPT sex determination for identical twins is exceptionally high, comparable to the accuracy seen in singletons.
Fraternal (Dizygotic) Twins
Fraternal twins result from two separate eggs and sperm, making them genetically distinct, like siblings. They can be two boys, two girls, or a mixed-sex pair. This distinction introduces complexity because the NIPT result aggregates two different DNA profiles.
While overall sex classification accuracy in twin pregnancies is high (sometimes reported around 98%), this figure masks the difficulty in identifying the sex of each twin individually in a mixed-sex pair. If NIPT detects a Y chromosome, it confirms at least one twin is male, but cannot distinguish between a male/male pair and a male/female pair. The highest accuracy occurs when no Y chromosome is detected, reliably confirming both twins are female.
What a Gender Result Means for Twins
The NIPT result for a twin pregnancy generally falls into one of two categories, reflecting the practical implications of mixed DNA analysis.
Y Chromosome Detected (Male Result)
A result indicating the presence of a Y chromosome confirms that at least one fetus is male. This outcome conclusively establishes that the pair is not two females. However, the test cannot distinguish between a male/male pair and a male/female pair, providing only partial information regarding the individual sexes of fraternal twins.
No Y Chromosome Detected (Female Result)
The absence of a Y chromosome signal is the most reliable outcome in twin NIPT. This result indicates that both fetuses are female, assuming the overall fetal fraction was sufficient. This conclusion is highly accurate because even a small amount of Y chromosome DNA from a male twin would typically be detected.
Follow-up confirmation is often recommended due to NIPT’s limitations in distinguishing mixed-sex pairs. High-resolution ultrasound, typically performed around 18 to 20 weeks of gestation, remains the standard method for visually confirming the sex of each twin. Genetic counseling ensures parents understand the probabilistic nature of the NIPT result and its limitations when a Y chromosome is detected.