First-trimester screening has become the cornerstone of prenatal risk assessment for chromosomal aneuploidies and certain structural malformations. The integration of sonographic measurement of nuchal translucency (NT) and maternal serum biochemical markers has substantially improved the early detection rates of conditions such as trisomy 21, trisomy 18, and trisomy 13.1,2 NT, defined as the sonographically measured subcutaneous fluid-filled space at the back of the fetal neck between 11 and 14 weeks of gestation, has consistently been recognized as one of the most powerful independent sonographic predictors of chromosomal and structural abnormalities. While an NT thickness of ≥3 mm is often considered the threshold for increased risk, its interpretation must be contextualized with maternal serum biomarkers to achieve optimal screening performance.3-5 Among the available biochemical markers, free β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein-A (PAPP-A), and alpha-fetoprotein (AFP) have been most extensively studied. Free β-hCG is a glycoprotein hormone produced by syncytiotrophoblasts and plays a pivotal role in trophoblastic proliferation and placental function. Altered levels of free β-hCG, particularly elevated concentrations, have been associated with an increased risk of trisomy 21 and adverse pregnancy outcomes.6,7 PAPP-A, a placental glycoprotein that regulates the availability of insulin-like growth factors, is typically reduced in pregnancies affected by chromosomal abnormalities and adverse obstetric outcomes.8 Similarly, AFP, synthesized primarily by the fetal liver, has traditionally been associated with neural tube defects but is increasingly recognized as a complementary marker in first-trimester screening when interpreted alongside NT and other serum parameters.9 The pathophysiological link between increased NT and aberrant serum marker levels is an area of ongoing investigation. Increased NT is thought to reflect impaired lymphatic drainage, cardiac dysfunction, or abnormalities in extracellular matrix composition, which may coincide with placental dysfunction reflected in altered serum biomarkers. Several studies have demonstrated that fetuses with enlarged NT not only carry higher risks of chromosomal abnormalities but may also show distinctive biochemical profiles, suggesting an overlapping mechanistic pathway.1,10 However, the strength and consistency of these associations vary across populations, and data from South Asian cohorts remain scarce. In regions such as Kashmir, where large-scale implementation of combined first-trimester screening is limited, there is a pressing need to generate population-specific data on the relationship between NT and serum biomarkers. Establishing these correlations may enhance the predictive accuracy of screening algorithms and improve counseling for pregnancies presenting with enlarged NT. Furthermore, such data can contribute to the refinement of risk stratification strategies, particularly in resource-constrained settings where invasive testing carries significant logistical and cultural barriers. The present study was designed to explore the association of NT thickness with maternal serum free β-hCG, PAPP-A, and AFP levels in fetuses identified with NT ≥ 3 mm during routine first-trimester screening. By focusing exclusively on the high-NT subset, we sought to clarify the relationship between abnormal sonographic findings and biochemical markers in a well-defined cohort. This approach not only provides insight into the pathophysiological interplay between placental function and fetal development but also has direct clinical implications for refining risk assessment in pregnancies with increased NT.

This prospective observational study was carried out over a period of 18 months in the Postgraduate Department of Anatomy in collaboration with the Departments of Obstetrics and Gynaecology and Radiodiagnosis and Imaging at the Government Medical College, Srinagar, Jammu and Kashmir. Pregnant women attending the antenatal clinic during the study period were screened for eligibility. Women with viable singleton pregnancies from 11 to 14 weeks of gestation, confirmed by crown–rump length measurement, were included, while those with gestational age less than 11 weeks or more than 14 weeks, multiple pregnancies, or refusal of consent were excluded. The study protocol was approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants prior to enrolment. For each woman, demographic and clinical details were recorded using a structured questionnaire. All participants underwent a transabdominal ultrasound examination using a LOGIQ P9 ultrasound system (General Electric, USA). Nuchal translucency thickness was measured following the guidelines of the Fetal Medicine Foundation by obtaining a mid-sagittal section of the fetus in a neutral position and measuring the maximum thickness of the subcutaneous translucency between the skin and soft tissue overlying the cervical spine. Three measurements were taken, and the largest satisfactory value was recorded. Women found to have a nuchal translucency measurement of 3 mm or more were identified as the study cohort for further biochemical evaluation. In these women, venous blood samples were obtained at the time of the ultrasound examination for maternal serum biochemical analysis. The biomarkers assessed included free β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein-A (PAPP-A, expressed as multiples of the median), and alpha-fetoprotein (AFP). All assays were carried out in the hospital’s central diagnostic laboratory using standardized immunoassay techniques. Women with increased NT were subsequently followed up throughout pregnancy to document outcomes, including the presence of chromosomal abnormalities and live birth status. The recorded data were compiled in Microsoft Excel and exported to SPSS version 23.0 (SPSS Inc., Chicago, Illinois, USA) for statistical analysis. Continuous variables were summarized as mean ± standard deviation, while categorical variables were presented as frequencies and percentages. Graphical representation was done using bar and pie charts. Comparisons between continuous variables were performed using Student’s independent t-test or the Mann–Whitney U test, depending on data distribution, while categorical variables were compared using the Chi-square test or Fisher’s exact test, as appropriate. Correlation between nuchal translucency thickness and serum biomarker levels was assessed using Pearson’s or Spearman’s correlation coefficients. A p-value of less than 0.05 was considered statistically significant.

A total of 22,679 women were screened between 11 and 14 weeks of gestation. Of these, 22 pregnancies (0.1%) were identified with nuchal translucency (NT) ≥ 3 mm and constituted the study cohort for biomarker analysis. Among the 22 fetuses with nuchal translucency (NT) ≥ 3 mm, the mean NT thickness was 4.36 ± 0.61 mm with a median of 4.25 (range: 3.40–5.40 mm). Table 1: Baseline ultrasound and biochemical characteristics of fetuses with NT ≥ 3 mm Parameter Mean SD Median Range Maximum Minimum Nuchal translucency (mm) 4.36 0.61 4.25 2.00 5.40 3.40 Fetal heart rate (bpm) 155.95 9.00 157.00 28.00 170.00 142.00 Crown–rump length (mm) 59.36 13.19 60.50 43.00 77.00 34.00 Gestational age (days) 89.18 11.02 88.00 44.00 116.00 72.00 Serum free β-hCG (IU/L) 123.87 68.65 121.00 201.20 231.00 29.80 Serum PAPP-A (MoM) 57.25 59.73 44.70 286.80 302.00 15.20 Alpha-fetoprotein (ng/mL) 37.75 17.38 25.59 48.79 68.91 20.12 The mean fetal heart rate (FHR) was 155.95 ± 9.00 bpm, ranging from 142 to 170 bpm, with a median of 157 bpm. The mean crown–rump length (CRL) measured 59.36 ± 13.19 mm (range: 34.00–77.00 mm), while the gestational age at examination averaged 89.18 ± 11.02 days (approximately 12 weeks and 5 days), with a median of 88 days (range: 72–116 days). With respect to serum biomarkers, the mean free β-hCG level was 123.87 ± 68.65 IU/L (range: 29.80–231.00 IU/L), the mean PAPP-A concentration was 57.25 ± 59.73 MoM (median: 44.70; range: 15.20–302.00 MoM), and the mean alpha-fetoprotein (AFP) was 37.75 ± 17.38 ng/mL (median: 25.59; range: 20.12–68.91 ng/mL). The mean NT thickness was 4.36 ± 0.61 mm, while the mean maternal serum free β-hCG concentration was 123.87 ± 68.65 IU/L. Correlation analysis demonstrated no significant association between NT and β-hCG levels (r = 0.027, p = 0.904) (Table 2). The mean NT thickness was 4.36 ± 0.61 mm, while the mean maternal serum PAPP-A level was 57.25 ± 59.73 MoM. Correlation analysis revealed a weak positive relationship between NT and PAPP-A, which did not reach statistical significance (r = 0.111, p = 0.622) (Table 3). Table 2. Correlation between nuchal translucency and maternal serum free β-hCG Parameter Nuchal Translucency Serum β-hCG (IU/L) Mean 4.364 123.868 SD 0.611 68.647 N 22 – Correlation coefficient (r) 0.027 – p-value 0.904 – Result Not significant – Table 3: Correlation between nuchal translucency and maternal serum PAPP-A (MoM) Parameter Nuchal Translucency Serum PAPP-A (MoM) Mean 4.364 57.254 SD 0.611 59.728 N 22 – Correlation coefficient (r) 0.111 – p-value 0.622 – Result Not significant – Table 4: Correlation between nuchal translucency and maternal serum alpha-fetoprotein Parameter Nuchal Translucency Alpha-fetoprotein (ng/mL) Mean 4.364 37.745 SD 0.611 17.375 N 22 – Correlation coefficient (r) 0.156 – p-value 0.486 – Result Not significant The mean NT thickness was 4.36 ± 0.61 mm, while the mean maternal serum AFP concentration was 37.75 ± 17.38 ng/mL. A weak positive correlation was observed between NT and AFP, but this association was not statistically significant (r = 0.156, p = 0.486) (Table 4).

In this prospective observational study, we assessed the association of increased nuchal translucency (NT ≥ 3 mm) with maternal serum biomarkers. Out of a total of 22,679 screened pregnancies, only 22 fetuses (0.1%) were identified with NT ≥ 3 mm and formed the focus of our biochemical evaluation. Among the 22 fetuses identified with enlarged NT, we observed mean NT of 4.36 ± 0.61 mm, which falls within the range generally considered “increased” at the 11–14 week scan, the standard window in which NT is measured with CRL 45–84 mm criteria aligned with FMF technique recommendations.11,12 The accompanying ultrasound parameters were consistent with the expected first-trimester range, with a mean crown–rump length of 59.4 mm and a mean gestational age of 12 weeks and 5 days, both aligning closely with established norms recommended in current first-trimester screening guidelines.12 Likewise, the mean FHR of 156 bpm (SD 9) lies toward the higher end of the accepted first-trimester physiologic range typically ~120–160 bpm with a known peak near 170 bpm around 9–10 weeks indicating that the cohort’s cardiovascular physiology was appropriate for gestational age.13,14 In our cohort of 22 fetuses with NT ≥ 3 mm, we observed mean levels of free β-hCG: 123.87 IU/L; PAPP-A: 57.25 MoM (median 44.7); and AFP: 37.75 ng/mL (median 25.6). When contextualized against reference ranges in typical first-trimester pregnancies, these values appear within expected limits for non-aneuploid, healthy gestations. For example, maternal AFP levels in the early second trimester typically fall between 10–150 ng/mL, and levels rise through the end of the first trimester, our findings are comfortably within this continuum.15These observations suggest that in pregnancies with increased NT, the mean maternal serum biomarkers may not exhibit extreme abnormalities; instead, NT elevation itself remains the most prominent early screening anomaly. In our series of fetuses with increased nuchal translucency (NT ≥ 3 mm), the mean NT thickness was 4.36 ± 0.61 mm, yet no significant associations were found between NT and the maternal serum biomarkers free β-hCG, PAPP-A, and AFP. Specifically, the mean maternal free β-hCG concentration was 123.87 ± 68.65 IU/L, but correlation analysis yielded no meaningful relationship with NT (r = 0.027, p = 0.904). Similarly, although the mean maternal PAPP-A level was 57.25 ± 59.73 MoM, only a weak positive correlation with NT was observed (r = 0.111, p = 0.622). For AFP, the mean value was 37.75 ± 17.38 ng/mL, with correlation analysis again demonstrating only a weak and statistically insignificant positive association with NT (r = 0.156, p = 0.486). This pattern is biologically plausible once cases are pre-selected by a high NT threshold: the ultrasound marker itself becomes the dominant risk signal, and incremental association with biochemistry tends to attenuate. In large population studies of first-trimester combined screening, low PAPP-A and high free β-hCG are characteristic of trisomy 21 (with both low in trisomies 18/13), and adding NT to serum markers markedly improves detection; yet these relationships are clearest in unselected cohorts rather than within the high-NT stratum where risk is already enriched by the sonographic phenotype.6,16-18 The pathophysiological mechanisms underlying NT enlargement also provide an explanation for this lack of association. Increased NT is thought to result from transient cardiac dysfunction, abnormal lymphangiogenesis (such as jugular lymphatic sac distension), and/or alterations in the extracellular matrix mechanisms that do not necessarily disrupt placental secretion of PAPP-A or hCG. Both prospective and review studies highlight the role of disturbed lymphatic development, and its timing relative to NT enlargement, in fetuses with and without structural heart disease, across both aneuploid and euploid cases. 19-21 This explains why NT can be substantially increased even when maternal serum markers remain within typical ranges. In this context, the absence of a clear trend in mean free β-hCG and mean PAPP-A across NT thickness aligns with prior reports indicating that risk at the population level is driven primarily by marker severity rather than average levels. Within cohorts selected for increased NT, serum biochemistry contributes relatively little additional information. Landmark modeling studies and multicenter trials have demonstrated that the strength of combined screening lies in the complementarity of independent markers across their full distributions. However, once NT is elevated (≥3 mm), the proportion of residual variance explained by serum analytes is markedly reduced.16,18 Regarding AFP, the first-trimester evidence base is mixed and generally weaker than for PAPP-A and hCG. Multiple studies conclude that first-trimester AFP adds little to aneuploidy screening; some contemporary analyses link elevated early AFP with placenta-mediated morbidity (FGR, preeclampsia, preterm birth), but not as an independent discriminator for chromosomal risk once standard markers are considered.22,23 The weak, non-significant correlation in our data therefore supports the consensus that AFP should not be considered a primary screening biomarker in the first trimester, particularly within high-NT pregnancies. It is pertinent to mention that observed associations may be weakened by restricted NT range, small sample size, unadjusted maternal and gestational covariates, and etiologic heterogeneity within the high-NT group.

The present study demonstrated that in pregnancies with nuchal translucency ≥ 3 mm, maternal serum free β-hCG, PAPP-A, and AFP do not correlate significantly with NT thickness. These findings suggest that, once NT is enlarged, it remains the principal determinant of risk, while the incremental contribution of biochemical markers becomes limited. Clinically, this underscores the role of increased NT as a critical trigger for comprehensive follow-up including detailed cardiac and anatomic assessment as well as genetic testing rather than relying on serum biochemistry to refine prognosis in this subgroup. Larger, multicenter studies with cytogenetic follow-up are warranted to clarify the nuanced interactions between NT and maternal serum markers and to strengthen evidence-based management pathways for high-NT pregnancies.

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