Integrating NIPT and ultrasound for detecting fetal aneuploidies and abnormalities

Clinical application of results

The detection of rare aneuploidies through NIPT combined with ultrasound markers demonstrates the strength of this integrated approach. These additional insights informed clinical decision-making, such as guiding parental counseling and determining the need for invasive procedures like amniocentesis. For cases with rare anomalies (e.g., suspected Trisomy 7 and 16), ultrasound findings such as severe growth restriction and cardiac abnormalities played a pivotal role in corroborating genetic results, enabling early diagnosis and tailored clinical management. This synergy is consistent with emerging data from recent studies that emphasize the improved risk stratification when both modalities are used concurrently [4], 5]. Such evidence highlights the importance of using both modalities to address limitations inherent in either method alone (Figure 6).

Figure 6:

This 4D ultrasound image displays facial features characteristic of Down syndrome, such as a flattened facial profile and midfacial hypoplasia; although precise measurement ranges are not provided, the image is intended to serve as a reference for clinicians in identifying these anomalies, with explanatory notes emphasizing the need for clinical correlation.

Diagnostic synergy and workflow implications

Integrating NIPT and ultrasound into diagnostic workflows provides a robust framework for prenatal care. NIPT offers genetic insights with high specificity, while ultrasound adds structural clarity, particularly in cases where cfDNA levels are insufficient. For example, in cases of Monosomy X, ultrasound markers such as increased NT and coarctation of the aorta provided diagnostic clarity when cfDNA signals were ambiguous. This dual-modality approach not only enhances diagnostic accuracy but also streamlines clinical pathways by reducing reliance on repeat testing or unnecessary invasive procedures. The integration of these modalities has been recently supported by several studies that demonstrate improved patient management and cost-effectiveness in prenatal screening protocols [1].

Ambiguity in cfDNA results and clinical management

Ambiguous cfDNA results, defined as cases with insufficient fetal fractions (<4 %) or inconclusive chromosomal findings, were observed in 4.21 % of cases. These ambiguous results posed a significant challenge, as they delayed definitive diagnoses and increased parental anxiety [1], 4]. In such cases, clinical management relied heavily on complementary ultrasound imaging to provide structural insights [7], 12]. For instance, ultrasound markers such as increased NT >3 mm or craniofacial anomalies played a pivotal role in clarifying suspected chromosomal anomalies [7], 12]. When ultrasound findings were inconclusive, repeat NIPT testing was performed – particularly when low cfDNA levels were attributed to early gestational age [16], 18], 19] – and invasive diagnostic procedures, such as amniocentesis, were offered when integrated findings suggested a high probability of chromosomal abnormalities [15], 18]. These management strategies are reinforced by recent literature that advocates for multimodal follow-up protocols to reduce diagnostic uncertainty [3].

Challenges and limitations

One key limitation of NIPT in these cases was its dependence on sufficient fetal fractions, which can be affected by factors such as maternal obesity, multiple pregnancies, or early gestational age [2], 3], 6], 8], 9]. These factors often necessitated additional testing, prolonging the diagnostic timeline. Moreover, ambiguous results occasionally failed to provide actionable insights, highlighting the need for enhanced diagnostic tools [16], 18]. As noted by Allyse et al., the variability in cfDNA yield remains a critical barrier to universal implementation, warranting further technological improvements and methodological refinements [11].

Future directions for managing ambiguities

To reduce ambiguity in cfDNA results, several strategies can be implemented. Expanding cfDNA panels to include microdeletions, duplications, and rare chromosomal abnormalities could provide a broader scope of analysis, minimizing inconclusive findings [12], 17]. Additionally, integrating AI into NIPT workflows could improve the interpretation of low fetal fractions by identifying and compensating for maternal factors affecting cfDNA levels [17], 18]. AI-powered systems can also predict the likelihood of cfDNA insufficiency, enabling clinicians to make informed decisions about repeat testing or alternative diagnostic methods. By leveraging these advancements and combining genetic data with detailed structural imaging, future prenatal diagnostic frameworks can offer more reliable and timely results, ultimately improving clinical outcomes and reducing parental stress. This perspective is increasingly supported by studies demonstrating that AI integration can enhance both the sensitivity and specificity of current screening methods [12], 16].

Clinical recommendations

Based on the findings from this study and the supporting literature, we propose the following clinical recommendations to optimize the integration of NIPT and ultrasound in prenatal diagnostics:

1. Initial Screening Protocols: NIPT should be offered to all pregnant women at or beyond 10 weeks of gestation, as it provides high sensitivity and specificity for detecting common aneuploidies such as Trisomy 21, 18, and 13 [1], [2], [3], [4]. For high-risk pregnancies (e.g., advanced maternal age, history of chromosomal abnormalities), NIPT should be combined with first-trimester ultrasound to assess critical markers such as NT [1], [2], [3], [4, 7], 12].

2. Follow-Up for Inconclusive NIPT Results: In cases where cfDNA levels are insufficient (<4 %), ultrasound should be prioritized to identify structural markers such as craniofacial anomalies or cardiac defects [4], 7]. Repeat NIPT testing may be considered if the cfDNA insufficiency is attributed to early gestational age or maternal obesity [8], 9].

3. Diagnostic Workflow for Detected Anomalies: When NIPT detects a high probability of chromosomal anomalies, follow-up ultrasound should confirm structural findings (e.g., holoprosencephaly in Trisomy 13 or overlapping fingers in Trisomy 18) to refine the diagnosis and guide counseling [12]. For ambiguous findings, such as suspected sex chromosome aneuploidies (e.g., Monosomy X), invasive testing (e.g., amniocentesis) may be warranted [12].

4. Diagnostic Algorithm: A stepwise diagnostic algorithm is recommended: Perform NIPT at 10 weeks or later. Follow up with ultrasound at 12–20 weeks to assess structural markers. Use integrated findings to guide the need for invasive testing.

5. Integration with Emerging Technologies: While this study focuses on current diagnostic modalities, incorporating advancements such as expanded cfDNA panels and AI-enhanced ultrasound holds promise for improving diagnostic accuracy in future applications.

The role of artificial intelligence (AI) in enhancing diagnostic accuracy

Findings and interpretations

This study confirmed that integrating NIPT with ultrasound imaging enhances the detection of chromosomal and structural anomalies. Among the 190 cases analyzed, NIPT achieved a cfDNA detection rate of 91.58 %, with aneuploidies identified in 4.74 % of cases, including Trisomy 21, 18, and 13, as well as Monosomy X and 47,XXX [1], 5]. Additionally, two cases of rare aneuploidies, Trisomy 7 and Trisomy 16, were flagged based on integrated findings (Figure 4). Ultrasound findings provided critical structural insights, particularly in ambiguous or overlapping cases. For instance, increased NT, observed in 70 % of Down syndrome cases (Table 1), was a significant marker that corroborated NIPT results [8]. Similarly, overlapping fingers and severe growth restriction were defining features in Edward syndrome (Figure 7; Table 2), while holoprosencephaly and craniofacial anomalies characterized Patau syndrome (Table 3). These structural markers, combined with genetic findings, allowed for more comprehensive and nuanced diagnoses.

Figure 7:

This Figure shows a 4D ultrasound image highlighting key features of Edwards syndrome, including a prominent occiput, micrognathia, and craniofacial asymmetry; while no standardized normal ranges exist for these features, the image offers a diagnostic reference and includes notes on their clinical relevance.

In cases where NIPT results were inconclusive or insufficient (8.42 % of cases; Figure 2), ultrasound played a pivotal role in refining diagnostic outcomes. Cardiac defects and skeletal abnormalities identified via imaging were particularly valuable in distinguishing genetic from non-genetic anomalies [12]. For example, microorchidism and borderline growth restriction in XXY syndrome (Table 4) provided diagnostic clarity in cases with normal karyotypes [12]. The combined diagnostic approach was especially impactful in rare or atypical cases, such as the suspected Trisomy 7 and Trisomy 16 cases. Here, ultrasound findings, such as severe growth restriction and cardiac anomalies, complemented NIPT data to provide a clearer diagnostic picture. This underscores the importance of a multi-modal approach in managing complex or overlapping findings. Recent literature supports these integrated methods as a means to reduce both false negatives and false positives in prenatal screening [6].

Figure 8:

This 4D ultrasound image reveals distinct craniofacial anomalies characteristic of Patau’s syndrome (Trisomy 13), offering valuable insights for prenatal diagnosis. Prominent features, such as holoprosencephaly (incomplete brain division), cleft lip or palate, and hypotelorism (close-set eyes), are visualized in high resolution, with the image serving as an illustrative guide for diagnosis. The advanced imaging allows for detailed assessment of facial malformations, which are key diagnostic markers of this condition. By providing a comprehensive view, this technique enhances early detection and enables better-informed clinical decisions and family counselling.

Case examples

This study highlights the practical implications of integrating NIPT with ultrasound imaging through several illustrative cases:

1. Case 1: Trisomy 21 (Down syndrome). A patient presented with NIPT results indicating a high probability for Trisomy 21. Follow-up ultrasound revealed hallmark markers such as increased NT >3 mm and the absence of a nasal bone, both observed in approximately 70 % of Down syndrome cases (Figure 6; Table 1). These combined findings facilitated early parental counselling and allowed the healthcare team to plan appropriate clinical management.

2. Case 2: Trisomy 13 (Patau syndrome). NIPT flagged a high risk for Trisomy 13, prompting a detailed ultrasound evaluation. Structural anomalies such as holoprosencephaly, polydactyly, and craniofacial malformations, including cleft lip and palate (Table 3), were confirmed through imaging (Figure 8). These findings corroborated the genetic results, enabling a definitive diagnosis and guiding the parents through informed decision-making.

3. Case 3: Isolated Structural Anomaly. A fetus with a normal karyotype presented with isolated cardiac defects, including a ventricular septal defect. These findings, detected via ultrasound, highlighted the importance of imaging in identifying non-genetic structural anomalies [12]. Clinical management focused on addressing the structural abnormality without the need for genetic interventions [12].

4. Case 4: Monosomy X (Turner syndrome). NIPT results for Monosomy X were inconclusive due to low cfDNA levels (<4 %). However, ultrasound findings such as increased NT and specific cardiac defects, including coarctation of the aorta, provided critical diagnostic clarity (Figure 2). This integrated approach enabled timely diagnosis despite limitations in cfDNA analysis [13].

5. Case 5: Ambiguous Fetal Sex Determination. In one case, low cfDNA levels led to ambiguous fetal sex determination. Ultrasound imaging clarified the issue by identifying normal male genital anatomy (Figure 9). This example underscores the ability of ultrasound to resolve diagnostic uncertainties when NIPT results are inconclusive [18].

These cases illustrate the synergistic power of combining NIPT with ultrasound imaging, particularly in cases with ambiguous or overlapping findings (Figure 9). The integrative approach not only enhances diagnostic precision but also enables personalized clinical care tailored to each patient’s unique circumstances.

Figure 9:

The 47 XXX syndrome, also known as Trisomy X, is a chromosomal condition that affects females and arises from the presence of an extra X chromosome in each cell. While many individuals with this condition have normal physical development and fertility, some may experience taller stature, learning disabilities, and delayed speech or motor skills. The condition is typically diagnosed through genetic testing, and most affected individuals lead healthy lives with appropriate support for any developmental challenges.

Sample limitations and representativeness

This study acknowledges several limitations in its sample size and demographic distribution, which may impact the generalizability of the findings. The cohort included 190 participants, predominantly drawn from urban and suburban regions, potentially excluding rural populations where access to prenatal care and diagnostic tools may differ significantly (Figure 5). Additionally, most participants were under 35 years of age (74.21 %), limiting the applicability of results to older maternal populations who are at a higher risk for chromosomal anomalies (Figure 1). Variability in socioeconomic factors, healthcare accessibility, and cultural attitudes toward prenatal testing were not fully accounted for, which could influence the study’s broader applicability [8]. Furthermore, the reliance on a single geographic location and the relatively small sample size may reduce the ability to detect less common chromosomal or structural anomalies [9]. Recent calls for larger, multicenter studies emphasize the need to include diverse populations to ensure more universally applicable findings [6].

Addressing limitations through AI

AI presents an innovative solution to address key limitations in prenatal diagnostics, such as operator variability in ultrasound imaging and challenges with NIPT [1], 4]. By standardizing the interpretation of ultrasound markers and enhancing the analysis of cfDNA data, AI-driven systems can improve diagnostic precision and consistency [1], 4]. Operator variability has long been a critical challenge in ultrasound diagnostics, impacting the detection of subtle anomalies such as NT irregularities or minor craniofacial malformations [12]. AI algorithms trained on large datasets of ultrasound images can mitigate these discrepancies by offering consistent and automated assessments. For instance, a study demonstrated that AI tools improved NT measurement accuracy by 95 % across operators, significantly reducing variability in results [20]. In the context of cfDNA analysis, AI has the potential to enhance fetal fraction detection, particularly in cases where low cfDNA levels have traditionally led to inconclusive results [9]. Emerging AI-based platforms are exploring ways to optimize cfDNA preprocessing and data interpretation, addressing challenges linked to maternal obesity and early gestational age [10], 11]. Such advancements could reduce the need for repeat testing and accelerate diagnosis, alleviating stress for expectant parents [12].

Recent advances in prenatal diagnostics

Recent original research has further refined integrated prenatal diagnostic approaches. Lin et al., introduced a novel algorithm that enhances NIPT’s prediction accuracy for Turner syndrome (45,X) by mitigating reference bias – a development that could reduce false-positive rates and improve clinical confidence [20]. Complementing this, Laporte et al., evaluated intrinsic fetal airway obstruction (CHAOS) through a rigorous correlation of ultrasound, fetoscopic, and pathological findings, underscoring the pivotal role of multi-modal imaging in accurately characterizing complex fetal structural anomalies [21]. In addition, Allen et al., demonstrated that clinically informed strategies significantly enhance the diagnostic yield for fetal structural anomalies in cases where conventional microarray testing remains non-diagnostic [22]. Collectively, these studies reinforce the trend toward integrating advanced genetic methodologies with detailed imaging and clinical data to achieve earlier, more precise, and personalized prenatal care.

Limitations and uncertainties in NIPT and ultrasound diagnostics

While NIPT has revolutionized prenatal diagnostics with its high sensitivity and specificity for common chromosomal anomalies, it is not without limitations. The reliance on sufficient cfDNA levels, often impacted by factors like maternal obesity, early gestational age, or multiple pregnancies, can lead to inconclusive results in 8.42 % of cases (Figure 2) [1], 4]. Low cfDNA levels necessitate repeat testing, delaying diagnosis and causing additional stress for expectant parents [5], 6]. NIPT is also limited in its ability to detect all genetic anomalies. For example, conditions such as balanced translocations, single-gene disorders, or some microdeletions may remain undetected due to the restricted scope of current cfDNA panels [8], 9]. As a result, cases with structural abnormalities identified through ultrasound but with normal karyotypes often require additional invasive procedures, such as amniocentesis, for definitive diagnosis [18].

Ultrasound imaging, while indispensable for identifying structural markers like NT and craniofacial anomalies, also has its challenges. Operator variability can significantly affect the accuracy of measurements, leading to inconsistencies in detecting subtle features such as minor skeletal abnormalities or cardiac defects [12]. Additionally, the timing of ultrasound assessments plays a crucial role; certain abnormalities may not be apparent until later stages of pregnancy, potentially delaying diagnosis and intervention [12]. Both NIPT and ultrasound face limitations when dealing with rare or overlapping conditions. For example, in cases of suspected Trisomy 7 or Trisomy 16, standalone results from either modality may yield insufficient clarity, requiring a multi-modal approach to achieve a reliable diagnosis [12]. Furthermore, socioeconomic factors and healthcare disparities can affect access to advanced diagnostic tools, creating inequities in prenatal care [19]. To address these challenges, advancements such as expanding cfDNA panels to include microdeletions and duplications and incorporating AI-driven ultrasound analysis are promising solutions. AI can standardize measurements, reducing operator variability and improving the detection of subtle anomalies such as ductus venosus abnormalities or borderline growth restrictions [20]. Furthermore, novel techniques to enhance cfDNA analysis, including pre-processing methods to improve fetal fraction detection, are under investigation and could significantly enhance NIPT reliability [17], 18]. By acknowledging and addressing these limitations, future research and clinical practices can refine prenatal diagnostics to ensure earlier, more accurate, and equitable outcomes for diverse patient populations [17], [18], [19], [20].

Implications of cfDNA insufficiency

The issue of insufficient cfDNA presents a significant challenge in the application of NIPT, particularly for populations with higher maternal BMI, early gestational age, or multiple pregnancies. In cases where cfDNA levels fall below the necessary threshold (4 %), as seen in 8.42 % of cases in this study (Figure 2), the reliability of NIPT results is compromised, necessitating repeat testing and often leading to delays in diagnosis [1], 4]. This insufficiency can be particularly problematic for women with high BMI, where the concentration of fetal cfDNA in maternal blood may be diluted, making it more difficult to achieve conclusive results [5], 6]. Moreover, in multiple pregnancies, cfDNA from each fetus may be indistinguishable, leading to diagnostic ambiguity and challenges in accurate fetal sex determination or chromosomal abnormality identification [8], 9]. In these cases, NIPT may not provide clear results, increasing the need for follow-up tests, including more invasive procedures like amniocentesis, to confirm the diagnosis [12].

The implications of cfDNA insufficiency extend beyond diagnostic delays. Inaccurate or ambiguous results can lead to unnecessary stress and anxiety for expectant parents, particularly when repeat tests are required or when results suggest the possibility of a chromosomal abnormality but cannot definitively confirm it [10]. The uncertainty surrounding cfDNA insufficiency is a critical issue that requires attention in order to improve the overall patient experience. To mitigate the impact of cfDNA insufficiency, researchers are exploring ways to enhance fetal fraction detection through novel techniques and preprocessing methods that could improve cfDNA analysis, especially in challenging populations such as women with higher BMI or in multiple pregnancies [11]. Additionally, combining NIPT with ultrasound imaging provides a complementary approach, enabling clinicians to rely on structural markers, such as NT or craniofacial anomalies, to confirm or clarify genetic findings [12]. Furthermore, the integration of AI into the NIPT process could improve the detection of low fetal fractions, offering more precise data interpretation and reducing the likelihood of inconclusive results [18]. AI-driven tools can also help standardize ultrasound assessments, ensuring consistent and accurate results even in cases where cfDNA levels are insufficient [20]. Addressing cfDNA insufficiency is essential for the continued improvement of NIPT. By refining technologies and integrating multi-modal approaches, prenatal diagnostics can become more reliable and accessible for all expectant parents, reducing the need for invasive testing and ensuring earlier, more accurate diagnoses.

Overall, these findings validate the efficacy of integrating genetic and structural diagnostics in prenatal care. The enhanced diagnostic precision achieved through combining NIPT with ultrasound imaging not only facilitates early, accurate detection of chromosomal and structural anomalies but also informs tailored clinical management strategies. As the field advances, further integration of emerging technologies – particularly AI – and expanded cfDNA panels are anticipated to overcome current limitations, ultimately improving outcomes for mothers and their children.

Clinical applications and implementation guidelines

The integration of NIPT and ultrasound imaging in prenatal diagnostics should follow a systematic algorithm that begins with offering NIPT at or after 10 weeks of gestation, ensuring that cfDNA levels meet the required threshold, and is followed by a detailed ultrasound examination between 12 and 20 weeks to assess key structural markers. This coordinated approach, which involves a multidisciplinary team – including obstetricians, genetic counselors, and certified sonographers – facilitates the prompt identification and confirmation of chromosomal and structural anomalies, thereby enhancing patient counseling and clinical management.

To ensure the highest diagnostic accuracy, it is imperative to implement rigorous quality assurance measures, such as standardized operating procedures, regular equipment calibration, and routine audits with peer reviews. In addition, establishing comprehensive training programs that include initial certification, periodic re-certification, and dedicated workshops on AI integration will help reduce operator variability and maintain high standards in both ultrasound and cfDNA analysis.

From a resource perspective, significant investments in state-of-the-art ultrasound equipment, reliable NIPT platforms, and AI software are necessary. Coupled with robust information technology (IT) infrastructure to support data integration and analysis, these resources can ultimately reduce the need for invasive procedures and minimize repeat testing, making the integrated diagnostic strategy not only clinically effective but also cost-efficient.