Aneuploidy Screening Notes

Introduction

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Aneuploidy Screening: A Vital Topic

Aneuploidy screening is vital both for postgraduate entrance exams and clinical practice. Aneuploidy refers to defects in chromosome number. Normally, humans have 46 chromosomes. Aneuploidy occurs when there are 45 or 47 chromosomes.

Down Syndrome

Down syndrome is trisomy 21, meaning there's an extra chromosome 21, resulting in 47 chromosomes.

Other Trisomies

Trisomy 18 is Edward syndrome, where there's an extra chromosome 18. Patau syndrome is trisomy 13, indicating an additional chromosome 13.

Monosomy

Monosomy means one less chromosome. Monosomy X (Turner syndrome) involves one less X chromosome, written as 44 + XO or 45,XO.

Aneuploidy Screening: Purpose

Aneuploidy screening checks for trisomies or monosomies in pregnant females. It should be offered universally, regardless of maternal age. The primary goal is to detect trisomy 21 (Down syndrome) and trisomy 18 (Edward syndrome).

Screening Test Results

If a screening test is positive, the next step is a diagnostic test like karyotyping. Karyotyping involves examining the fetus's complete chromosomal makeup using fetal tissue.

Karyotyping

A normal karyotype shows 22 pairs of chromosomes and sex chromosomes (XX for female, XY for male). In trisomy 21, there are three copies of chromosome 21, resulting in a total of 47 chromosomes.

Approaches After Positive Screening

After a positive screening test, there are two approaches:

1. Direct karyotyping (invasive).
2. Secondary non-invasive screening with cell-free fetal DNA in maternal blood. If this is positive, proceed to karyotyping.

Cell-Free Fetal DNA Test

The cell-free fetal DNA test is expensive and may not be affordable for all patients. Some patients may opt for direct karyotyping.

Special Cases: Previous Trisomy

If a pregnant female has a history of a previous baby with Down syndrome or any trisomy, skip the screening test and directly perform a diagnostic test (karyotyping).

Screening Methods

Aneuploidy screening can be done in the first trimester, second trimester, or both.

First Trimester Screening

Methods include biochemical markers and ultrasound, or a combination of both.

Biochemical Markers: Dual Test

The dual test involves measuring PAPPA (pregnancy-associated plasma protein A) and beta hCG levels in the mother's blood.

• In Down syndrome, hCG levels are high, while PAPPA levels are decreased.
• In Edward syndrome, both hCG and PAPPA levels are decreased.

Interpreting Ratios

Reports provide a risk ratio. For Down syndrome, the cutoff is 1 in 250; for Edward syndrome, it's 1 in 100. If the risk is higher than the cutoff, the screening is positive.

Examples

• Down syndrome risk of 1 in 300 is a negative screening (lower risk).
• Down syndrome risk of 1 in 100 is a positive screening (higher risk).

Ultrasound: Nuchal Translucency

Ultrasound measures nuchal translucency (fluid-filled area below the skin in the neck region).

Guidelines for Measurement

• Measured in the midsagittal plane.
• Fetal head should be in a neutral position.
• Head, neck, and thorax should be magnified.
• Amnion should be seen separately.
• Measure from inner border to inner border at the widest plane.

Best Time

The best time to measure nuchal translucency is between 11 weeks to 13 weeks plus 6 days, or when the crown-rump length is between 45 to 84 mm.

Interpretation

If nuchal translucency is $\geq$ 3mm, it indicates potential problems like trisomy (most common), Turner syndrome, or cardiac defects. After increased nuchal translucency, karyotyping is performed. If the karyotype is normal, echocardiography is recommended to check for cardiac defects.

Cystic Hygroma

Cystic hygroma is a more widespread fluid collection, often with septa. It's a stronger marker for aneuploidy than increased nuchal translucency.

• In the first trimester, it's most commonly associated with Down syndrome.
• In the second trimester, it's most commonly associated with Turner syndrome.

Normal Karyotype with Cystic Hygroma

If karyotyping is normal but cystic hygroma is present, consider congenital heart diseases (hypoplastic left heart or coarctation of the aorta), genetic syndromes like Noonan syndrome, or hydrops fetalis. Echocardiography is recommended.

Combined Test

Combined test includes both the dual test and nuchal translucency measurement for increased sensitivity in detecting aneuploidy (85%).

Positive Screening Test

If the screening test is positive, the confirmatory test is karyotyping, requiring fetal tissue obtained via chorionic villi biopsy in the first trimester.

Second Trimester Screening

Done between 15 to 20 weeks, ideally between 16 to 18 weeks.

Biochemical Methods

Triple Test

Triple test (or Kettering test/Bart test) is now outdated. It checks for alpha-fetoprotein, hCG, and unconjugated estriol (uE3).

• In Down syndrome: AFP decreased, hCG increased, uE3 decreased.
• In Edward syndrome: All three are decreased.

Sensitivity is 70%.

Quad Test

Quad test includes the triple test markers plus inhibin A. In Down syndrome, inhibin A levels are increased. Sensitivity is 80%, and this is the preferred test.

Ultrasound

Looks for soft tissue markers. The presence of any two markers indicates a positive screening for Down syndrome or aneuploidy.

Soft Tissue Markers

• Nuchal skin fold thickness ( $\geq$ 6mm)
• Absent nasal bone
• Short humerus
• Short femur
• Simian crease
• Sandal gap
• Echogenic cardiac focus
• Echogenic bowel
• Pelvic calcium dilatations
• Choroid plexus cyst

Isolated Findings

Nuchal skin fold thickness as an isolated finding has the highest risk of aneuploidy, followed by a short femur. Choroid plexus cyst has the lowest risk.

Integrated Screening

It combines first and second-trimester screenings. It involves dual test and nuchal translucency in the first trimester, and quad test in the second trimester.

Biochemical Markers

Integrated screening checks five biochemical markers (hCG, PAPPA, AFP, uE3, and inhibin A) plus nuchal translucency.

Sensitivity

Integrated screening is more sensitive than first or second-trimester screening alone.

Important

Do not confuse combined screening (dual test + nuchal translucency) with integrated screening (dual test + quadruple test + nuchal translucency).

Positive Second Trimester Screening

Confirmatory test is karyotyping, with fetal tissue obtained via amniocentesis.

Non-Invasive Prenatal Test (NIPT)

NIPT involves taking a sample of the mother's venous blood to check for cell-free fetal DNA. The source of this DNA is the placenta.

• Sensitivity: 99%
• Detects: Trisomy 21, 18, 13, and Turner syndrome
• Disadvantages: Expensive, limited to detecting only these four chromosomal problems, and still requires confirmatory karyotyping if positive
• Timing: Any time beyond 10 weeks of pregnancy

Prenatal Genetic Diagnostic Tests

These are confirmatory tests that provide a complete chromosomal makeup of the fetus.

Tests

• Karyotyping (most common).
• FISH (fluorescent in situ hybridization) for quick results.

Fetal Tissue

Fetal tissue can be obtained by:

• Chorionic villi sampling.
• Amniocentesis.
• Cordocentesis.

Chorionic Villi Sampling vs. Amniocentesis

Cordocentesis

It involves taking samples from the umbilical vein. High fetal loss rate (2-3%). Not used for genetic testing due to high risk.

Procedure

Sampling occurs from the umbilical vein near the placental end.

Time Frame

Performed at $\geq$ 18 weeks.

Uses

Used to determine the exact hemoglobin or hematocrit of the fetus, especially in cases of fetal anemia (e.g., Rh isoimmunization) when peak systolic velocity in the middle cerebral artery is >1.5 MOM.

Important One-Liners

Down Syndrome Cause

The most common cause of Down syndrome is nondisjunction of chromosomes.

Recurrence Risk

If a previous Down syndrome baby had nondisjunction, the recurrence risk is 1%. If it was due to balanced translocation of chromosomes, the recurrence risk is 100%.

Prenatal vs. Preimplantation Genetic Testing

• Prenatal genetic testing involves karyotyping with material obtained via chorionic villi sampling or amniocentesis.
• Preimplantation genetic testing is done in case of IVF, checking zygotes for health before implantation using polar bodies or blastocysts.