Residency · Residency · Medical Genetics Genomics
Invasive Prenatal Diagnosis: Amniocentesis and CVS
Overview
Amniocentesis and chorionic villus sampling (CVS) are the two primary invasive procedures for obtaining fetal tissue for genetic diagnosis. Both provide definitive diagnostic results, in contrast to screening tests such as cfDNA, serum screening, and ultrasound. ACOG recommends that diagnostic testing be offered to all pregnant individuals regardless of age or risk status. The choice between procedures depends on gestational age, indication, and available laboratory testing.
Chorionic Villus Sampling (CVS)
Technique
CVS is performed at 10-13 weeks gestation, optimally at 11-12 weeks. It samples chorionic villi (placental tissue) via either a transcervical approach using a catheter guided by ultrasound through the cervix, or a transabdominal approach using a needle inserted through the abdominal wall into the placenta under ultrasound guidance. Route selection depends on placental location, uterine position, and operator experience. Typically 10-25 mg of villi are obtained.
Laboratory Processing
Two preparations are performed. Direct preparation analyzes actively dividing cytotrophoblast cells and produces results in 1-2 days, though it reflects the cytotrophoblast layer and may not represent the fetus. Cultured preparation grows mesenchymal core cells for 7-14 days and is more representative of the fetus. Both preparations are recommended for complete analysis, and discrepancies between them raise concern for mosaicism.
Advantages
CVS offers earlier diagnosis in the first trimester, allowing earlier decision-making. Results are available sooner in gestation, and the procedure yields better-quality DNA for some molecular testing compared to amniocytes. It is preferred when rapid karyotype or molecular results are needed early.
Limitations
CVS cannot assess amniotic fluid (for AFP or acetylcholinesterase), carries risk of confined placental mosaicism, is not available after 13 weeks (14 at some centers), and carries somewhat higher risk of maternal cell contamination than amniocentesis.
<image>Illustration comparing transcervical and transabdominal CVS techniques with ultrasound guidance, showing needle/catheter placement relative to placenta, and the distinction between cytotrophoblast and mesenchymal core cell layers in chorionic villi</image>
Amniocentesis
Technique
Amniocentesis is performed at 15-20 weeks gestation (typically 15-18 weeks). An ultrasound-guided transabdominal needle is inserted into the amniotic cavity, and 20-30 mL of fluid is withdrawn (approximately 1 mL per week of gestation). The fluid contains fetal cells (amniocytes shed from fetal skin, urinary tract, respiratory tract, and GI tract) along with cell-free components.
Laboratory Processing
Cultured cells require 7-14 days of amniocyte growth for karyotyping. FISH provides rapid results within 24-48 hours for common aneuploidies (chromosomes 13, 18, 21, X, Y) using uncultured cells. Chromosomal microarray can be performed on uncultured cells with faster turnaround of 5-7 days. DNA extraction enables sequencing, methylation studies, or targeted molecular assays. Biochemical analysis of the supernatant assesses AFP and acetylcholinesterase for neural tube defects, and enzymatic assays can diagnose lysosomal storage disorders and other metabolic conditions.
Advantages
Amniocentesis carries lower risk of confined placental mosaicism compared to CVS because amniocytes are fetal rather than placental in origin. Amniotic fluid is available for biochemical analysis. The procedure is widely available and can be performed later in pregnancy for late-arising indications.
Limitations
Later gestational timing (15+ weeks) delays results compared to CVS. Low-level mosaicism in cultured amniocytes may represent either true fetal mosaicism or culture artifact (pseudomosaicism). Early amniocentesis before 14 weeks is not recommended due to increased risk of talipes equinovarus and pregnancy loss.
Procedural Risks
Pregnancy Loss
The traditionally quoted risk of approximately 1/200 to 1/300 for both procedures has been revised downward by contemporary data from experienced centers, which suggest procedure-attributable loss rates of approximately 1/500 to 1/1,000. The procedure-attributable rate is inherently difficult to determine because the background loss rate at 10-16 weeks is naturally elevated. CVS and amniocentesis appear to carry similar procedure-related loss rates when performed by experienced operators. Factors increasing risk include operator inexperience, multiple needle insertions, and uterine anomalies.
Other Complications
Amniotic fluid leakage occurs in approximately 1-2% of amniocentesis cases, with most sealing spontaneously. Infection (chorioamnionitis) occurs in fewer than 1 in 1,000 cases. RhoGAM (anti-D immunoglobulin) must be administered to Rh-negative unsensitized women after both procedures. Fetal injury is extremely rare with ultrasound guidance. Preterm labor and premature rupture of membranes are rare complications.
<image>Side-by-side comparison of amniocentesis and CVS procedures showing timing, technique, sample obtained, available laboratory tests, turnaround time, and risk profiles in a summary table format</image>
Confined Placental Mosaicism (CPM)
Definition and Incidence
CPM describes a chromosomal abnormality present in the placenta but not in the fetus. It is found in approximately 1-2% of CVS samples and represents the major source of discordant results between CVS and fetal karyotype. CPM does not occur in amniocentesis because amniocytes are fetal in origin.
Types of CPM
Type I involves mosaicism confined to the cytotrophoblast (direct preparation abnormal, culture normal). Type II involves mosaicism confined to the mesenchymal core (direct preparation normal, culture abnormal). Type III involves mosaicism in both cytotrophoblast and mesenchyme but not in the fetus. Type III is the most clinically concerning because it may be associated with uniparental disomy (UPD) in the fetus.
Clinical Significance
CPM involving chromosomes with imprinted genes (chromosomes 6, 7, 11, 14, 15, 20) warrants evaluation for UPD in the fetus. UPD can cause disease even when the fetal karyotype is normal, such as UPD15 causing Prader-Willi or Angelman syndrome. CPM may also be associated with adverse pregnancy outcomes (intrauterine growth restriction, preeclampsia) due to placental dysfunction. Follow-up amniocentesis is recommended when CVS reveals mosaicism to clarify fetal chromosomal status.
| Feature | CVS | Amniocentesis |
|---|---|---|
| Timing | 10–13 weeks | 15–20 weeks |
| Sample | Chorionic villi (placental) | Amniotic fluid (fetal cells) |
| Technique | Transcervical or transabdominal | Transabdominal |
| Cell preparations | Direct (cytotrophoblast) + cultured (mesenchyme) | Cultured amniocytes + uncultured for FISH/CMA |
| Results turnaround | 1–2 days (direct); 7–14 days (culture) | 24–48 hr (FISH); 5–7 days (CMA); 7–14 days (culture) |
| Biochemical analysis | Not available (no amniotic fluid) | AFP, AChE for NTDs; enzyme assays |
| CPM risk | ~1–2% (major limitation) | Very low (amniocytes are fetal) |
| Procedure-related loss | ~1/500–1/1,000 | ~1/500–1/1,000 |
| Key advantage | Earlier diagnosis (first trimester) | Lower CPM risk; biochemical testing available |
Testing Options on Prenatal Samples
Karyotype
Karyotype remains the gold standard for detecting numerical and large structural chromosome abnormalities, with a resolution of approximately 5-10 Mb. It requires cultured cells with 7-14 day turnaround and can detect balanced translocations and inversions that chromosomal microarray cannot.
Chromosomal Microarray (CMA)
CMA is recommended as the first-line test for pregnancies with structural anomalies on ultrasound per ACOG/SMFM guidelines. It detects submicroscopic deletions and duplications (CNVs) beyond karyotype resolution, providing an additional diagnostic yield of approximately 6% over karyotype in fetuses with structural anomalies and approximately 1.7% even with normal ultrasound. CMA cannot detect balanced rearrangements or low-level mosaicism below 20-30%. Variants of uncertain significance (approximately 2-4% of cases) create counseling challenges.
FISH
FISH enables rapid aneuploidy detection for chromosomes 13, 18, 21, X, and Y on uncultured cells with results in 24-48 hours. It does not replace full karyotype or CMA but provides valuable preliminary results.
Exome and Genome Sequencing
Prenatal exome sequencing is increasingly offered for fetuses with structural anomalies and normal karyotype/CMA. Diagnostic yield is approximately 8-10% for isolated anomalies and 15-20% for multiple anomalies after normal karyotype and CMA. Trio analysis (fetus plus both parents) is preferred for optimal interpretation. Challenges include VUS, incidental findings, turnaround time, and cost.
Targeted Molecular Testing
Single-gene testing, gene panels, or methylation studies are performed when a specific condition is suspected, such as FGFR3 testing for suspected skeletal dysplasia, SMA carrier testing for at-risk couples, or methylation testing for Beckwith-Wiedemann syndrome.
<image>Algorithm for selecting appropriate genetic testing in prenatal diagnosis based on clinical indication, including first-tier testing with karyotype or CMA, and escalation to exome sequencing when initial testing is nondiagnostic</image>
Indications for Invasive Prenatal Diagnosis
Indications include a positive or high-risk screening result (cfDNA, serum screening, nuchal translucency), fetal structural anomaly on ultrasound, known parental chromosomal rearrangement, known carrier status for a single-gene disorder, previous child with a chromosomal abnormality or genetic condition, advanced maternal age (though no longer a sole indication per ACOG), parental anxiety or desire for definitive information, and abnormal first-trimester screening findings.
Clinical Pearls
cfDNA is a screening test while CVS and amniocentesis are diagnostic tests -- this distinction is paramount and must be clearly communicated to patients. When CVS reveals mosaicism, follow-up amniocentesis is recommended to determine the true fetal karyotype. UPD testing should always be considered when CPM involves an imprinted chromosome, especially chromosomes 7, 11, 14, and 15. CMA is recommended over karyotype as the primary test when a fetal structural anomaly is detected on ultrasound. "Early amniocentesis" before 14 weeks is contraindicated due to increased complication rates. Rh-negative women must receive RhoGAM after any invasive procedure. Prenatal exome sequencing should be performed as trio and includes pre-test counseling about VUS and secondary findings. Cell culture failure occurs in approximately 0.5-1% of amniocentesis samples, but CMA on uncultured cells can still provide results.
References
- ACOG Practice Bulletin No. 162: "Prenatal diagnostic testing for genetic disorders." Obstetrics & Gynecology. 2016;127(5):e108-e122.
- Wapner RJ, Martin CL, Levy B, et al. "Chromosomal microarray versus karyotyping for prenatal diagnosis." New England Journal of Medicine. 2012;367(23):2175-2184.
- Grati FR, Malvestiti F, Ferreira JC, et al. "Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results." Genetics in Medicine. 2014;16(8):620-624.
- Lord J, McMullan DJ, Eberhardt RY, et al. "Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study." Lancet. 2019;393(10173):747-757.
- Akolekar R, Beta J, Picciarelli G, et al. "Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis." Ultrasound in Obstetrics & Gynecology. 2015;45(1):16-26.
