A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination.

Overview

• This study investigates various methods for preimplantation genetic testing (PGT) of equine embryos produced both in vivo and in vitro, focusing on determining the embryo’s sex.
• The research compares traditional biopsy methods with less invasive techniques and explores the potential for non-invasive analysis of genetic material shed by embryos into their culture medium.

Background and Objectives

• Preimplantation genetic testing (PGT) is commonly used in equine reproduction to determine genetic traits, including sex, before embryo implantation.
• Two primary PGT methods have been established for horse embryos:
  • Trophoblast Cell Biopsy (TCB) – extracting cells from the outer layer of the embryo (trophoblast).
  • Blastocoele Fluid Aspiration (BFA) – collecting fluid from the blastocoele cavity inside the embryo.
• While TCB is used for embryos produced both in vivo (inside the mare) and in vitro (in the lab), BFA has mainly been applied to in vivo-produced embryos.
• Newer, less invasive techniques such as analyzing cell-free DNA (CFD) released into the culture medium have been described in human PGT but have not yet been explored in horses.
• The study aims to evaluate and compare these methods for accuracy and safety in equine embryos, particularly for sex determination.

Experiment 1: Effects of Sequential Biopsy Procedures

• Objective: Assess if performing BFA followed by TCB 24 hours later impacts embryo development.
• Method:
  • Embryos produced in vivo (n=10) and in vitro (n=13) were first subjected to BFA.
  • They were cultured for 24 hours, then underwent TCB.
  • Post-biopsy, embryos were cultured an additional 24 hours.
• Results:
  • No significant changes in embryo size or ability to re-expand were observed following these procedures for either embryo group (p > 0.05).
  • This indicates that performing two biopsies one day apart does not harm embryo viability.

Experiment 2: Concordance of Sex Determination Among Techniques

• Objective: Evaluate the agreement in sex determination results using BFA, TCB, and analysis of the whole embryo.
• Method:
  • Performed PCR targeting sex-determining genes:
    • SRY and TSPY – genes on the Y chromosome indicating male sex.
    • Androgen receptor (AR) gene – present on the X chromosome.
  • Samples included those taken via BFA, TCB, and whole embryo lysates.
  • Analyzed concordance—the consistency of sex detection across methods.
• Results:
  • In vivo-produced embryos showed higher concordance (67-100%) among methods compared to in vitro-produced embryos (31-92%).
  • The target gene influenced concordance:
    • TSPY yielded better agreement rates (77-100%) than SRY (31-100%), suggesting TSPY is more reliable for sexing in this context.

Experiment 3: Analysis of Cell-Free DNA (CFD) in Culture Medium

• Objective: Test the feasibility of non-invasive sex determination using CFD present in the culture medium of in vitro-produced embryos.
• Method:
  • Collected culture medium samples surrounding in vitro-produced embryos.
  • Performed PCR targeting SRY (Y chromosome) and amelogenin (AMEL, present on both X and Y chromosomes) genes.
  • Compared results to sex determination from the whole embryo.
• Results:
  • CFD was detected in the culture medium of all embryos tested.
  • Concordance between CFD and whole embryo sex determination was 60% using these gene targets.
  • This indicates potential for a non-invasive PGT method, but accuracy needs improvement.

Conclusions and Implications

• It is safe to perform two biopsy procedures (BFA followed by TCB) on equine embryos 24 hours apart without detrimental effects on embryo size or viability.
• For in vivo-produced embryos, BFA can serve as a viable alternative to TCB for PGT, offering possibly less invasive sampling.
• For in vitro-produced embryos, BFA showed lower concordance and may be less reliable, so TCB remains the preferred biopsy method currently.
• Analysis of cell-free DNA from embryo culture medium represents a promising non-invasive approach for PGT in equine embryos, but accuracy remains below that of biopsy techniques and requires further refinement.
• Overall, this research helps improve genetic testing strategies in equine reproduction, optimizing embryo handling to support breeding programs and reduce invasive interventions.