Non-Invasive Prenatal Testing (NIPT) analyzes small fragments of cell-free DNA (cfDNA) found in a pregnant person’s bloodstream. These fragments originate from the placenta, which shares genetic material with the developing fetus. The test is primarily utilized to screen for common chromosomal conditions, such as Down syndrome, Edwards syndrome, and Patau syndrome. NIPT is also frequently used to determine fetal sex because it analyzes fetal genetic material early in pregnancy. This blood test offers a non-invasive alternative to traditional methods, but its capability to accurately determine the sex of both fetuses in a twin pregnancy presents unique technical and interpretive challenges.
The Mechanism of NIPT Sex Determination
NIPT determines the sex of a fetus by searching for the presence of the Y chromosome within the cfDNA sample. Genetic sex is determined by the combination of sex chromosomes inherited from the parents (XX for female, XY for male). The test specifically looks for sequences from the SRY gene, which is located on the Y chromosome and initiates male development.
If the analysis detects DNA sequences belonging to the Y chromosome, the result indicates the fetus is genetically male. Conversely, if no Y chromosome material is detected in the maternal blood sample, the result is interpreted as the fetus being genetically female. In a singleton pregnancy, this method provides a highly accurate determination of sex, often exceeding 99% accuracy.
Why Twin Pregnancies Complicate NIPT Accuracy
The primary complication in twin pregnancies arises because the cfDNA collected is a single, mixed sample containing the pregnant person’s DNA and the placental DNA from both fetuses. This combined genetic material makes it impossible to distinguish which DNA fragments belong to Fetus A and which belong to Fetus B.
The “fetal fraction,” the proportion of fetal cfDNA relative to the total cfDNA, is also combined in twins. While the total fetal fraction may be higher than in a singleton, the amount contributed by each individual twin can be inconsistent. Studies have shown that the circulating DNA contribution from one twin can be up to two times greater than the other. This unequal contribution means that one fetus’s genetic signature could dominate the sample, potentially obscuring the signal from the co-twin.
This mixed and unbalanced sample is the technical reason why NIPT cannot individually sex both fetuses with absolute certainty. If one twin contributes a very low amount of cfDNA, a result related to that twin, whether for sex or aneuploidy screening, may be less reliable or even inconclusive. Furthermore, a “vanishing twin,” where one fetus stops developing early in the pregnancy, can leave residual DNA fragments in the mother’s blood, leading to a potentially inaccurate or discordant NIPT result.
Interpreting Gender Results for Identical and Fraternal Twins
The interpretation of NIPT gender results in twins depends on the presence or absence of the Y chromosome and the zygosity of the fetuses (identical or fraternal). Two distinct scenarios emerge from the NIPT analysis, providing different levels of certainty.
Scenario 1: Y Chromosome Detected
When the NIPT detects the presence of the Y chromosome in the combined cfDNA sample, this confirms that at least one fetus is genetically male. If the twins are identical (monozygotic), they share the exact same genetic material, and the presence of Y DNA confirms both fetuses are male. If the twins are fraternal (dizygotic), the presence of the Y chromosome means the pair could be boy/boy or boy/girl. The test cannot differentiate between these two possibilities because the DNA from the two genetically distinct fetuses is mixed.
Scenario 2: No Y Chromosome Detected
The second scenario, where the NIPT detects no Y chromosome material, is the only result that provides certainty regarding the sex of both fetuses. The absence of Y DNA definitively concludes that both twins are female. This is why a “female” result is highly reliable for the sex of both twins, while a “male” result leaves an element of ambiguity for fraternal pairs. Some advanced NIPT platforms use sophisticated analysis to attempt to distinguish between the two fetal DNA profiles, which can sometimes provide individual sex determination for fraternal twins.