Residency · Residency · Medical Genetics Genomics

Preimplantation Genetic Testing (PGT-A, PGT-M, PGT-SR)

Overview

Preimplantation genetic testing (PGT) involves biopsy and genetic analysis of embryos created through in vitro fertilization (IVF) prior to transfer, allowing selection of embryos free from specific genetic conditions or chromosomal abnormalities before pregnancy is established. Three types exist: PGT-M for monogenic/single-gene disorders, PGT-SR for structural rearrangements, and PGT-A for aneuploidy. This terminology was updated in 2017 by PGDIS, replacing the earlier designations PGD and PGS.

Embryo Biopsy Techniques

Trophectoderm Biopsy (Day 5-7 Blastocyst)

Trophectoderm biopsy is the current standard of care, having replaced earlier biopsy stages. Five to ten cells are removed from the trophectoderm, the outer cell layer destined to become the placenta. This approach provides more cells for analysis (higher DNA yield) and has less impact on embryo development than cleavage-stage biopsy. Embryos are vitrified (flash-frozen) after biopsy pending results, then thawed for frozen embryo transfer in a subsequent cycle. A key limitation is that the trophectoderm may not perfectly represent the inner cell mass (ICM), which becomes the fetus -- a situation analogous to confined placental mosaicism.

Cleavage-Stage Biopsy (Day 3)

This older technique removed a single blastomere from a 6-8 cell embryo. It has been largely abandoned due to higher risk of embryo compromise, lower DNA yield, and higher mosaicism rates at this developmental stage.

Polar Body Biopsy

Polar body biopsy removes the first and/or second polar body from the oocyte. It only assesses the maternal genetic contribution and cannot detect paternally inherited or de novo mutations. Clinical use is limited, primarily restricted to countries where embryo biopsy is legally prohibited.

<image>Illustration of embryo biopsy techniques at different developmental stages: polar body biopsy from oocyte, cleavage-stage biopsy at day 3, and trophectoderm biopsy at blastocyst stage (day 5-7), with diagrams showing cells removed from each</image>

PGT-M: Testing for Monogenic Disorders

Indications

PGT-M is available for couples at risk for a known single-gene disorder, whether autosomal dominant, autosomal recessive, or X-linked. Examples include cystic fibrosis, sickle cell disease, Huntington disease, Marfan syndrome, spinal muscular atrophy, beta-thalassemia, and hereditary cancer syndromes (BRCA1/2, Lynch syndrome, Li-Fraumeni). HLA matching, with or without disease testing, can be performed for an existing affected sibling requiring hematopoietic stem cell transplantation (the "savior sibling" concept). PGT-M for late-onset conditions such as Huntington disease and BRCA1/2 is available but ethically debated.

Methodology

PGT-M requires pre-cycle workup (typically 6-8 weeks) to develop a customized testing protocol for the family. Linkage-based analysis uses informative STR or SNP markers flanking the gene of interest, tested across the family. Direct mutation analysis targets the specific pathogenic variants. Karyomapping is a genome-wide SNP linkage approach that simultaneously provides aneuploidy information and haplotype-based single-gene analysis. The combination of direct mutation detection plus linkage analysis is standard practice to reduce allele dropout risk.

Allele Dropout (ADO)

Allele dropout represents the failure to amplify one of two alleles during whole genome amplification of the few biopsied cells. ADO rates range from 2-10% per locus and can cause misdiagnosis if the pathogenic allele fails to amplify (false negative) or the normal allele drops out (false positive). Linkage analysis with flanking markers mitigates this risk by providing redundant information. ADO is the primary source of diagnostic error in PGT-M.

PGT-SR: Testing for Structural Rearrangements

Indications

PGT-SR is indicated when one partner carries a balanced chromosomal rearrangement, including reciprocal translocations, Robertsonian translocations, inversions, and complex rearrangements. Carriers of balanced rearrangements have high rates of unbalanced gametes leading to implantation failure, miscarriage, or offspring with congenital anomalies.

Methodology

Array-based or NGS-based analysis of trophectoderm biopsy detects unbalanced products of meiotic segregation (duplications/deletions of translocated segments). PGT-SR cannot distinguish balanced carrier embryos from structurally normal embryos, as both appear "balanced/normal." SNP array-based methods can potentially make this distinction using haplotype information if family samples are available. Embryos are classified as normal/balanced (suitable for transfer), unbalanced (not transferred), or aneuploid (not transferred if concurrent PGT-A is performed).

Reproductive Outcomes for Translocation Carriers

Without PGT-SR, 50-70% of embryos are unbalanced, leading to high miscarriage rates. PGT-SR improves pregnancy rates and reduces miscarriage risk. Robertsonian translocation carriers may have approximately 75% of embryos abnormal (monosomic or trisomic for involved chromosomes).

PGT-A: Testing for Aneuploidy

Rationale

Aneuploidy is the leading cause of implantation failure, miscarriage, and IVF failure. Aneuploidy rates increase with maternal age, with over 50% of embryos aneuploid in women over 38 years. PGT-A aims to select euploid embryos for transfer to improve IVF success rates.

Methodology

Current platforms use NGS-based analysis (gold standard) or array-based analysis of trophectoderm biopsy, assessing all 24 chromosomes for whole-chromosome and segmental gains and losses. Results are reported as euploid, aneuploid, mosaic, or no result. The goal is single euploid embryo transfer to reduce multiple pregnancy rates.

Mosaicism Detection

NGS platforms can detect chromosomal mosaicism (a mixture of euploid and aneuploid cells in the biopsy), reported when aneuploidy level falls between approximately 20-80% (thresholds vary by laboratory). Prevalence is approximately 5-20% of biopsied blastocysts. Mosaic embryos present a counseling challenge: some can result in healthy live births as aneuploid cells may be selected against during development. Transfer of mosaic embryos may be considered when no euploid embryos are available, following a priority hierarchy from euploid to low-level mosaic to high-level mosaic to complex mosaic. Prenatal diagnosis via amniocentesis is recommended if a mosaic embryo transfer results in pregnancy.

<image>Chart showing the relationship between maternal age and embryo aneuploidy rate, overlaid with the distribution of euploid, aneuploid, and mosaic embryos detected by PGT-A across different age groups</image>

Controversy: Does PGT-A Improve IVF Outcomes?

Arguments in favor include reduced time to pregnancy by avoiding transfer of aneuploid embryos, reduced miscarriage rate per transfer, confident single embryo transfer reducing twin risk, and greatest benefit in women of advanced maternal age with higher aneuploidy rates. Arguments against include no clear improvement in cumulative live birth rate per IVF cycle in large randomized controlled trials, the possibility that trophectoderm biopsy may not accurately reflect ICM ploidy (discarding potentially viable embryos), evidence that some aneuploid embryos may self-correct, added cost and complexity, potential reduction of total embryos available for transfer, and inconsistent results from the STAR trial and other RCTs on intention-to-treat analysis.

Current professional guidance reflects this controversy. ASRM/SART state that PGT-A may be offered but should not be considered mandatory, with patients counseled about limitations. ESHRE takes a more cautious stance, recommending PGT-A primarily in research settings. PGDIS supports PGT-A with appropriate counseling about mosaicism management.

Ethical Considerations

PGT-A reveals chromosomal sex, making nonmedical sex selection possible though controversial and prohibited in many countries. PGT-M for late-onset diseases like Huntington disease, BRCA1/2, and Alzheimer disease raises questions about severity thresholds. HLA typing for "savior siblings" raises concerns about commodification. PGT for conditions such as deafness or achondroplasia generates debate about what constitutes a "disorder." Embryo disposition (discard, research donation, indefinite storage) is another ethical dimension. Nondisclosure PGT for Huntington disease, where the at-risk parent does not wish to know their own status, requires a complex protocol.

<image>Comparison table of PGT-M, PGT-SR, and PGT-A showing indications, methodology, pre-cycle workup requirements, turnaround time, accuracy, and key limitations for each type</image>

Clinical Pearls

Trophectoderm biopsy reflects placental tissue, not the fetus directly, and mosaicism discordance between trophectoderm and ICM is a recognized limitation analogous to CPM in CVS. PGT-M requires extensive pre-cycle workup with family samples, so referral should occur months before the planned IVF cycle. Allele dropout is the Achilles heel of PGT-M, making linkage analysis with flanking markers essential for diagnostic accuracy. PGT-A cannot distinguish balanced translocation carrier embryos from structurally normal embryos without additional haplotyping. Prenatal diagnosis via CVS or amniocentesis is recommended for all PGT pregnancies as confirmatory testing, especially after mosaic embryo transfer. Mosaic embryo transfer decisions should involve genetic counseling, discussion of potential outcomes, and a plan for prenatal follow-up. PGT-A does not test for single-gene disorders, structural rearrangements (unless large), or epigenetic abnormalities. The cumulative live birth rate per egg retrieval cycle may not be improved by PGT-A -- this nuance is critical for informed consent.

References

• Practice Committee of the ASRM and the Society for Assisted Reproductive Technology. "The use of preimplantation genetic testing for aneuploidy (PGT-A): a committee opinion." Fertility and Sterility. 2020;114(2):246-272.
• Vermeesch JR, Voet T, Devriendt K. "Prenatal and pre-implantation genetic diagnosis." Nature Reviews Genetics. 2016;17(10):643-656.
• Gleicher N, Patrizio P, Brivanlou A. "Preimplantation genetic testing for aneuploidy -- a castle built on sand." Trends in Molecular Medicine. 2021;27(8):731-742.
• Spinella F, Fiorentino F, Biricik A, et al. "Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments." Fertility and Sterility. 2018;109(1):77-83.
• ESHRE PGT Consortium Steering Committee. "ESHRE PGT Consortium good practice recommendations for the organisation of PGT." Human Reproduction Open. 2020;2020(3):hoaa021.

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