Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences

Area of Science:

• Reproductive biology
• Human genetics
• Molecular diagnostics

Background:

• Preimplantation genetic diagnosis (PGD) is crucial for preventing the transmission of X-linked genetic disorders.
• Accurate sex determination of human embryos is essential for PGD strategies targeting X-linked conditions.
• Existing methods face challenges in reliability and accuracy when analyzing single cells.

Purpose of the Study:

• To develop and validate dependable methods for preimplantation sex determination of human IVF embryos.
• To enhance diagnostic accuracy for clinical applications in families at high risk of X-linked diseases.
• To minimize misdiagnosis by addressing potential pitfalls in single-cell genetic analysis.

Main Methods:

• Development of a multiplex nested polymerase chain reaction (PCR) protocol for rapid analysis of single cells and blastomeres.
• Co-amplification of X and Y chromosome amelogenin gene segments and Y-linked DYZ1 repetitive elements for enhanced reliability.
• Individual cell isolation and washing to prevent contamination, coupled with internal controls for error detection.

Main Results:

• The optimized single-cell protocol achieved a sexing error rate of less than 2% (1 in 60 samples).
• Multiplex amplification and internal controls effectively identified and excluded aberrant samples, averting misdiagnosis.
• The method demonstrated concordant sex determination in human triploid embryos and was adapted for blastocyst biopsy analysis.

Conclusions:

• Dependable and accurate preimplantation sex determination methods for human IVF embryos have been established.
• The developed protocol significantly reduces diagnostic errors, offering a reliable tool for PGD in at-risk families.
• The findings highlight the potential and address the challenges of single-cell genetic analysis in clinical settings.