Genetic Testing Confirms Monozygosity in Dichorionic Diamniotic Twins

A single case report published in Frontiers in Cell and Developmental Biology describes a dichorionic diamniotic twin pregnancy in which postnatal genetic testing confirmed monozygosity. The study, led by researchers from Universidad UTE in Ecuador, highlights a clinical scenario where standard ultrasonographic findings suggested a discrepancy in fetal development that could not be resolved by imaging alone. By utilizing short tandem repeat profiling, the team demonstrated that the twins shared a single zygote origin, challenging the common clinical assumption that dichorionic placentation invariably indicates dizygotic conception.

Twin pregnancies are clinically classified by chorionicity and amnionicity, which are critical factors for risk assessment. Dichorionic diamniotic twins, in which each fetus has its own chorion and placenta, are most frequently dizygotic, resulting from the fertilization of two independent oocytes. However, a small proportion of these pregnancies are monozygotic, a fact that complicates the interpretation of prenatal findings. In this specific case, a 34-year-old nulliparous woman presented with a twin gestation where early ultrasound examinations revealed a seven to nine-day discrepancy in estimated gestational age between the fetuses.

This initial discrepancy raised clinical concerns for several conditions, including selective fetal growth restriction, asynchronous conception, or the rare phenomenon of superfetation. Superfetation involves a second fertilization and implantation occurring during an ongoing pregnancy, leading to embryos of different developmental ages. The clinical team noted that while marked first-trimester crown-rump length discordance has been associated with adverse outcomes, the predictive accuracy of these measurements alone is limited. The underlying cause of such early intertwin differences can often be attributed to measurement variability, differences in implantation timing, or biological variation in embryonic growth.

To monitor the pregnancy, the medical team performed serial obstetric ultrasound examinations throughout all trimesters. Despite the initial difference in estimated gestational age, both fetuses exhibited concordant growth patterns. Intertwin discordance remained within normal limits, with less than a 10% difference in crown-rump length during the first trimester and less than a 25% difference in estimated fetal weight in subsequent evaluations. These longitudinal assessments effectively excluded selective fetal growth restriction, as the growth trajectories remained parallel without clinically significant biometric divergence.

Delivery occurred by cesarean section at 35 weeks and 4 days of gestation due to imminent preterm premature rupture of membranes. Both neonates had favorable outcomes and nearly identical anthropometric measurements at birth. The final prenatal ultrasound showed highly similar biometric parameters, with Twin A having an estimated fetal weight of 2,005.72 grams and Twin B at 2,133.1 grams. These measurements were consistent with appropriate growth for the estimated gestational age, further supporting the absence of persistent intertwin growth divergence during the pregnancy.

To determine the zygosity of the twins, the researchers performed postnatal genetic analysis using standard forensic methodology. Buccal samples were collected from both newborns using the Whatman FTA EasyCollect system. The DNA was processed and assessed for quantity and quality before undergoing short tandem repeat amplification. The GlobalFiler PCR Amplification Kit was used to target 21 autosomal STR loci, along with amelogenin for sex determination and Y-chromosomal markers. This approach is considered the gold standard for zygosity determination due to the high polymorphism and well-characterized inheritance patterns of these genetic markers.

The STR analysis revealed complete genetic concordance across all successfully amplified loci. No differences were detected between the genetic profiles of the two twins. Identical genotypes were observed at all analyzed informative autosomal markers, including D3S1358, vWA, and D16S539. Both samples also showed matching sex chromosome markers, with identical results for amelogenin and Y-chromosomal markers confirming the male sex of both newborns. This complete concordance strongly supports a monozygotic origin, indicating that both twins share the same genetic profile for the analyzed markers.

The findings illustrate that chorionicity alone does not determine zygosity. While the classical timing model of monozygotic twinning suggests that dichorionic twins result from early embryonic separation, the relationship between separation timing and chorionicity is more complex. DCDA monozygotic pregnancies have been reported following the transfer of a single blastocyst, demonstrating that dichorionic development can occur despite the embryo having reached the blastocyst stage. The present case cannot establish the specific mechanism of embryonic separation, but it reinforces the need to interpret the classical timing model as a general conceptual framework rather than an invariant biological rule.

The study acknowledges several limitations inherent to a single-case report. The findings cannot be generalized to other DCDA twin pregnancies or used to establish the frequency of such presentations. Furthermore, the timing and cellular mechanism of embryonic separation could not be directly investigated, and placental histopathological information was not available. Although STR profiling provides strong molecular evidence for monozygosity, it evaluates only a limited fraction of the genome. Higher-resolution genomic approaches, such as whole-genome sequencing, might detect genomic differences not captured by conventional STR analysis.

From a clinical perspective, chorionicity and zygosity should be regarded as related but distinct biological characteristics. Chorionicity reflects placental and membrane organization and is fundamental for defining prenatal surveillance, whereas it does not necessarily establish whether twins originate from one or two zygotes. Molecular determination of zygosity is not routinely required for all DCDA pregnancies, as obstetric management is primarily guided by chorionicity and fetal clinical findings. However, zygosity testing may provide additional information in selected situations where biological origin remains uncertain or has implications for clinical interpretation, such as in cases with discordant biometric findings or when superfetation is considered.

The integration of longitudinal ultrasonographic findings with postnatal genetic assessment provided complementary information for interpreting this clinically discordant presentation. The case demonstrates the value of molecular zygosity testing in resolving ambiguities that arise when chorionicity and early biometric findings do not align. By confirming monozygosity in a dichorionic diamniotic pregnancy, the study underscores the importance of genetic methods in understanding the developmental mechanisms of twinning and clarifying the biological relationship between twins in complex clinical scenarios.

This report contributes to the broader understanding of twin embryology and the limitations of imaging-based inference. It highlights that while ultrasonography is indispensable for assessing chorionicity and fetal growth, it may not always resolve questions related to biological origin. The use of STR profiling in this context offers a reliable method for determining zygosity, which can assist in the interpretation of discordant findings that cannot be attributed to different genetic backgrounds. The study serves as a reminder that clinical classifications based on placental number must be interpreted with caution, particularly when early gestational age discrepancies are present.

Beyond the primary biometric assessments, maternal ultrasound imaging revealed specific anatomical features that initially complicated the diagnostic evaluation. An examination of the ovaries identified a well-vascularized corpus luteum within the right ovary, whereas no corresponding structure could be detected in the left ovary. This unilateral ovarian finding pointed toward potential visualization limits of pelvic imaging or raised the possibility of asynchronous ovulation. Additionally, sonographic examination detected a cervical hematoma, though clinicians confirmed that this collection did not involve either gestational sac. Serial biometric surveillance recorded parallel progression across mid-gestation: at approximately 21 weeks, Twin A had an estimated fetal weight of 472.97 grams compared to 369.31 grams for Twin B, alongside biparietal diameters of 5.05 and 4.70 centimeters, before converging at roughly 28 weeks to 1,132.01 grams and 1,145.39 grams.

The forensic genetic evaluation adhered to rigorous laboratory protocols to verify sample integrity and genotyping accuracy. After extracting biological material from the collection cards, researchers utilized the Quantifiler Trio DNA Quantification Kit to evaluate DNA concentration and purity. Capillary electrophoresis of the amplified products was conducted using a 3500 Genetic Analyzer, followed by data interpretation with GeneMapper ID-X software. Laboratory quality control incorporated Control DNA 007 as a positive amplification control to validate successful primer binding and allele calling, alongside a no-template reaction as a negative control to exclude laboratory contamination. The resulting profiles demonstrated complete matching across all tested autosomal loci, including TPOX, D21S11, D18S51, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, and D2S1338, as well as the Y-chromosomal marker DYS391 and the Y-indel.

The forensic reliability of short tandem repeat markers stems from their high polymorphism and predictable Mendelian inheritance. For autosomal markers, individuals inherit one allele from each parent, producing a biparental genotype that offers exceptional discriminatory power for relationship testing and personal identification. In sex chromosome analysis, X-linked markers follow maternal inheritance in males, while Y-linked markers pass intact through the paternal lineage. These properties have established STR testing as a standard method in forensic science for identifying human remains, investigating missing persons, resolving criminal casework, assessing organ transplantation matching, and estimating biological kinship.

Embryologically, chorionic configuration depends on the differentiation of distinct early cell lineages. The outer trophectoderm primarily gives rise to the chorion and placental structures, whereas the pluripotent inner cell mass develops into the embryo itself. Under classical twinning models, embryonic splitting prior to the morula stage separates both lineages to create dichorionic twins. Clinical management frameworks reflect these biological complexities; guidelines from the International Society of Ultrasound in Obstetrics and Gynecology recommend specialist referral for significant intertwin discordance and advise formal zygosity testing during invasive genetic evaluations, especially in same-sex dichorionic pairs presenting with discordant structural or biometric findings.

Subject of Research: Determination of zygosity in dichorionic diamniotic twin pregnancies using short tandem repeat profiling

Article Title: Genetics meets embryology: redefining zygosity in a dichorionic diamniotic twin pregnancy through STR profiling

Article References: Cadena-Ullauri, S., Ruiz-Pozo, V. A., Tamayo-Trujillo, R., Guevara-Ramírez, P., Paz-Cruz, E., Meza-Chico, L., López-Carrera, M., Rivadeneyra-Alban, C., Llerena Béjar, L. I., & Zambrano, A. K. (2026). Genetics meets embryology: redefining zygosity in a dichorionic diamniotic twin pregnancy through STR profiling. Frontiers in Cell and Developmental Biology, 14, Article 1857119. https://doi.org/10.3389/fcell.2026.1857119

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1857119

Keywords: dichorionic diamniotic twins, monozygosity, zygosity determination, short tandem repeat profiling, twin pregnancy, fetal growth discordance, ultrasonographic assessment, embryonic development, chorionicity, forensic genetics, superfetation, selective fetal growth restriction, genetic concordance, obstetric ultrasound, twin-to-twin transfusion, placental architecture, neonatal outcomes, genetic markers, twin embryology, clinical genetics