Pre-Implantation Techniques - Clinical Notes

Overview

1. The IVF Framework (Prerequisite)

• Ovarian stimulation - to retrieve approximately 10 or fewer oocytes per cycle
• In vitro fertilization of retrieved oocytes
• Embryo culture for 5-6 days until the blastocyst stage (day 5 or 6)
• Embryo biopsy, genetic analysis, then transfer (fresh or frozen-thaw cycle)

2. Biopsy Techniques - How Cells Are Sampled

A. Polar Body Biopsy

• Timing: At the oocyte stage, before or after fertilization (very early)
• Method: The first and/or second polar body is removed and analyzed; the genetic status of the oocyte is inferred indirectly
• Limitation: Crossing over during meiosis can cause erroneous diagnosis. Critically, it only reveals the maternal contribution - it cannot differentiate a carrier from an affected embryo for recessive disorders

B. Blastomere Biopsy (Day 3 / Cleavage Stage)

• Timing: Eight-cell stage (day 3 embryo)
• Method: One or two blastomeres are mechanically removed and analyzed by PCR
• Limitations:
  • Only 1-2 cells available = very limited DNA
  • Risk of allele dropout - preferential amplification of one allele over the other
  • Sperm DNA contamination from zona pellucida can cause false-negatives
  • High rate of mosaicism at cleavage stage means a single blastomere may not represent the whole embryo's karyotype (trisomic rescue means aneuploid cells at this stage may not persist)
• This method underperformed in a large multicenter RCT (2003-2007, n=408): PGT-A using FISH on cleavage-stage blastomeres gave a lower pregnancy rate (25% vs 37%) compared to standard IVF - Creasy & Resnik's Maternal-Fetal Medicine

C. Trophectoderm Biopsy (Blastocyst Stage - Current Standard)

• Timing: Day 5 or 6, at blastocyst stage
• Method: 5-10 cells are removed from the trophectoderm (which becomes the placenta) without disturbing the inner cell mass (ICM, which becomes the fetus)
• Advantages over blastomere biopsy:
  1. More DNA available (5-10 cells vs 1-2 cells) = greater diagnostic accuracy
  2. Less damaging to the embryo
  3. Less mosaicism overall at this stage compared to cleavage stage
  4. ICM is left entirely intact
• Limitation: Either rapid genetic testing within 24 hours is needed OR embryos must be cryopreserved for a frozen-thaw transfer (freeze-all strategy)

3. The Three Types of PGT

4. PGT-A (Aneuploidy Testing)

Rationale

• Women >35 years have >30% aneuploid embryos; this rises to ~60% by age 40
• Selecting euploid embryos theoretically improves implantation and live birth rates

Evolution of Technology (Version 1.0 → 2.0)

• PGT-A 1.0: FISH probes on cleavage-stage blastomeres - tested only a limited set of chromosomes; no longer used due to poor clinical outcomes
• PGT-A 2.0 (current): Trophectoderm biopsy + genome-wide methods:
  • Array CGH (comparative genomic hybridization) - previously the gold standard
  • Quantitative PCR
  • Massive Parallel Sequencing (Next Generation Sequencing / NGS) - now the most common; same principle as exome/genome sequencing; early evidence it may also detect clinically significant mosaicism

ASRM Position

Limitations of PGT-A

• Cannot detect subchromosomal deletions/duplications (only whole chromosome)
• Cannot detect whole chromosome mosaicism, triploidy, or single-gene disorders
• False-negative rate for microarray PGT-A: <1%
• False-positive/negative overall: ~2-3% - confirmatory prenatal testing (CVS or amniocentesis) still recommended
• Does not replace standard prenatal screening; cfDNA screening should still be offered
• The biopsy site (trophectoderm) may not perfectly represent the ICM karyotype
• Sources of error: sample mislabeling, DNA contamination, amplification failure, array noise, trophectoderm-ICM karyotype discordance

5. PGT-M (Monogenic Testing)

• PCR amplification of the specific variant
• Analysis of linked polymorphic markers (STRs, SNPs) is included alongside direct variant detection to:
  • Improve accuracy
  • Detect allele dropout
  • Minimize misdiagnosis from sperm contamination
• SNP microarrays after whole genome amplification (WGA) can overcome allele dropout
• Confirmatory CVS or amniocentesis is recommended after successful transfer because of the ~2-3% false result rate

6. PGT-SR (Structural Rearrangements)

• Used when one or both parents carry a balanced chromosomal translocation or other structural rearrangement
• Detects unbalanced rearrangements in embryos
• Uses the same NGS/array platforms as PGT-A; SNP arrays also allow detection of unbalanced translocations
• Prevents implantation of embryos with unbalanced chromosomal products, which often lead to miscarriage or affected offspring

7. Special Consideration - Mosaicism

• Mosaicism: Some cells in an embryo are euploid, others are aneuploid (due to mitotic errors during early development)
• A significant challenge because the trophectoderm biopsy sample may not represent the ICM
• Mosaic embryos can be considered potentially viable but are deprioritized for transfer compared to non-mosaic euploid embryos
• Prioritization for mosaic embryo transfer:
  • Single mosaic chromosome involvement > two or more chromosomes
  • Lower degree of aneuploidy (fewer affected cells) is preferred over higher degree
• Approximately 100 documented live births after mosaic embryo transfer with no significant adverse events reported (as of available data) - Creasy & Resnik

8. Genetic Technologies Used in PGT

9. Summary of Biopsy Methods Comparison

10. Clinical Indications for PGT

• Advanced maternal age (>35 years) - PGT-A
• Recurrent pregnancy loss (RPL) - PGT-A
• Repeated IVF implantation failure - PGT-A
• Known carrier of Mendelian disorder (CF, SMA, sickle cell, thalassemia, BRCA, etc.) - PGT-M
• Carrier of chromosomal translocation or inversion - PGT-SR
• Sex-linked disorders (X-linked recessive conditions) - PGT-M or sex selection
• Neuropsychiatric disorders: Fragile X, Huntington disease, Alzheimer disease risk - PGT-M (Kaplan & Sadock's Comprehensive Textbook of Psychiatry)
• Mitochondrial DNA disorders (selective cases)

• Creasy & Resnik's Maternal-Fetal Medicine: Principles and Practice, blocks covering PGT sections
• Thompson & Thompson Genetics and Genomics in Medicine, 9th ed.
• Berek & Novak's Gynecology
• Emery's Elements of Medical Genetics and Genomics