Prenatal Screening for Trisomy 21: Fetal Medicine Approach

Introduction

1. Risk Stratification: Maternal Age

• Age-related risk table (Creasy & Resnik MFM, p. 646): at 12 weeks, risk ranges from 1:1068 (age 20) to 1:49 (age 45)

2. First-Trimester Biochemical Screening (10-13+6 weeks)

• PAPP-A alone: detection rate (DR) ~40-45% at 5% false-positive rate (FPR)
• Free β-hCG alone: DR ~23% at 5% FPR
• Combined (PAPP-A + free β-hCG): DR 60-65% at 5% FPR
• Total hCG can substitute but is slightly less discriminating than free β-hCG

3. First-Trimester Ultrasound Markers

Nuchal Translucency (NT)

• Measured by a Fetal Medicine Foundation (FMF)-accredited sonographer using strict quality criteria
• NT alone achieves a DR of ~75-80% at 5% FPR
• Must be expressed as a MoM or delta-NT corrected for crown-rump length

Combined First-Trimester Test (FTS)

• DR: 82-87% at 5% FPR (the standard first-trimester approach)

Additional First-Trimester Ultrasound Markers (can adjust risk further)

• Absent nasal bone: present in ~60-70% of T21 fetuses at 11-13 weeks (only ~3% of euploid fetuses)
• Reversed ductus venosus flow (absent/reversed a-wave)
• Tricuspid regurgitation on Doppler
• Frontomaxillary facial angle: increased in T21

4. Second-Trimester Biochemical Screening ("Quad Screen"): 15-20 weeks

5. Combined Integrated and Sequential Screening Strategies

• 1st-trimester combined test result stratifies patients into three groups:
• High risk (>1:30): offered immediate invasive testing
• Intermediate risk (1:30-1:1300): proceed to 2nd-trimester quad screen
• Low risk (<1:1300): no further screening needed (screen complete)
• Only 15-20% proceed to 2nd-trimester testing
• DR: 92-94% at 5% FPR with greatest cost-efficiency

6. Second-Trimester Ultrasound Soft Markers

7. Cell-Free DNA (cfDNA) / Non-Invasive Prenatal Testing (NIPT)

Biology

• 3-15% of circulating maternal plasma DNA is cell-free fetal/placental DNA (cfDNA), derived primarily from placental trophoblast apoptosis
• cfDNA consists of small fragments (<450 bp) with a half-life of ~16 minutes
• Available from 9 weeks' gestation until delivery
• Fetal fraction must be adequate (typically ≥4%) for a reportable result

Methods

1. Massively parallel (shotgun) sequencing (MPS): All maternal plasma DNA is sequenced; chromosome 21 fragments are counted relative to disomic chromosomes. A fetus with T21 produces ~1.05x more chr 21 fragments when fetal fraction is 10%.
2. Targeted massive parallel sequencing: Focuses on specific chromosomes of interest
3. Single-nucleotide polymorphism (SNP)-based analysis: Distinguishes maternal vs fetal alleles and can detect triploidy and uniparental disomy

Performance (Trisomy 21)

• Sensitivity: >99%
• Specificity: >99.9%
• False-positive rate: ~0.1%
• PPV is population-dependent (higher in high-risk populations; lower in general population)
• In low-risk populations, PPV for T21 ~45-90% depending on prevalence - so a "positive" cfDNA still requires confirmatory invasive testing

Current Position in Guidelines (ACOG/SMFM/RCOG)

• cfDNA is the most sensitive screening test for T21 available
• Offered as primary screening to all women (not restricted to high-risk)
• A positive cfDNA result requires confirmation by invasive diagnosis before pregnancy termination
• A no-result/failed cfDNA has increased risk for aneuploidy and warrants follow-up

8. Invasive Diagnostic Testing

• Positive/high-risk screening result (combined test, cfDNA, quad screen)
• Previous child with trisomy 21 (empiric recurrence risk ~1-1.5% if mother <30 at index pregnancy)
• Structural fetal anomaly on ultrasound
• Parental chromosomal rearrangement
• Patient preference for definitive answer regardless of screening

Chorionic Villus Sampling (CVS)

• Timing: 10-13 weeks
• Route: Transcervical or transabdominal under ultrasound guidance
• Procedure-related miscarriage risk: ~0.5-1% (some meta-analyses cite ~1% above background)
• Advantages: Earlier result (first trimester), allows earlier management decisions
• Pitfalls: Confined placental mosaicism (~1-2%); may require follow-up amniocentesis
• Samples can be sent for karyotype, chromosomal microarray (CMA), or QF-PCR

Amniocentesis

• Timing: ≥15 weeks (ideally 15-18 weeks)
• Route: Transabdominal under continuous ultrasound guidance
• Procedure-related miscarriage risk: ~0.1-0.5% above background (lower than CVS per updated meta-analyses)
• Advantage: Lower procedure risk, gold-standard for karyotype
• Early amniocentesis (<15 weeks) carries higher risk (clubfoot, failed culture) and is not recommended

What Diagnostic Tests Are Performed on Sample

• G-banded karyotype: Takes 2-3 weeks; detects all major aneuploidies and structural rearrangements
• QF-PCR (Quantitative fluorescent PCR): Rapid result (24-48h) for common aneuploidies (T21, T18, T13, sex chromosomes) - used for urgent preliminary result
• Chromosomal microarray (CMA): Detects copy number variants (CNVs) missed on karyotype; recommended over karyotype alone when structural anomaly is present
• Whole exome sequencing (WES): Emerging role when microarray is negative but structural anomalies are present

Summary Table: Screening Tests and Their Performance

Key Points for Exam

1. cfDNA is screening, NOT diagnostic - a positive result always requires invasive confirmation before definitive action
2. The combined first-trimester test (NT + PAPP-A + free β-hCG + age) remains the standard where cfDNA is unavailable or unaffordable
3. Absent nasal bone, reversed DV flow, and tricuspid regurgitation are powerful additional 1st-trimester markers that refine risk
4. PPV of any screening test depends on prevalence in the population screened
5. CVS vs amniocentesis: CVS is earlier (allows 1st-trimester diagnosis) but carries slightly higher procedural risk and risk of confined placental mosaicism; amniocentesis is the gold standard with slightly lower risk
6. All women should be offered both screening AND diagnostic testing options regardless of age (ACOG/SMFM 2020 guidance)