Non-invasive human preimplantation embryos sex determination using STR-based fluorescent multiplex PCR on days 3 and...

Area of Science:

• Reproductive Biology
• Genetics
• Molecular Diagnostics

Background:

• Preimplantation genetic testing (PGT) traditionally relies on invasive embryo biopsy.
• Non-invasive methods for PGT are highly desirable to improve safety and efficiency.
• Cell-free DNA (CF-DNA) in spent culture medium presents a potential source for non-invasive genetic analysis.

Purpose of the Study:

• To evaluate the efficacy of CF-DNA from spent culture medium (SCM) for sex identification using short tandem repeat (STR) markers.
• To compare the accuracy of CF-DNA sex identification with fluorescence in situ hybridization (FISH).
• To explore the potential of non-invasive PGT for sex determination and chromosomal abnormalities.

Main Methods:

• Collected spent culture medium (SCM) from human embryos at day 3 and day 5.
• Isolated CF-DNA from SCM and blastocoel fluid.
• Performed STR-based multiplex PCR for sex identification and compared results with FISH analysis.

Main Results:

• CF-DNA analysis from SCM demonstrated 100% specificity, sensitivity, positive, and negative predictive values for sex identification.
• The Agreement Kappa (AK) value between CF-DNA and FISH was 0.516, indicating moderate agreement.
• No correlation was found between allele dropout (ADO) and embryo morphology, but embryo morphology correlated with marker failure rates.

Conclusions:

• Embryonic CF-DNA in SCM is a viable source for sex identification, showing consistency with FISH.
• STR marker analysis of CF-DNA in culture medium offers a promising non-invasive approach for sex determination.
• Future advancements may enable non-invasive PGT for aneuploidy (PGT-A) and monogenic disorders (PGT-M) without embryo biopsy.