Isolation of Cell Free Fetal DNA From Spent Culture Medium and Its Potential Role for Preimplantation Genetic Diagnosis (PGD).
Condition: Females Undergoing PGD Technology for Medical Conditions
Study type: Observational

Summary: 
      Preimplantation genetic testing for chromosomal screening is, at present, the most reliable
      method to assess the genetic status of preimplantation embryos. DNA isolated and amplified
      from trophectoderm (TE) biopsiesis the method currently used for preimplantation genetic
      testing, but it has significant limitations. There is increasing evidence for a ''true''
      noninvasive approach consisting of the analysis of cell-free DNA (cfDNA) released by the
      embryo into the spent blastocyst medium (SBM) during the late stages of preimplantation
      development.

      This study is to assess whether Noninvasive prenatal genetic diagnosis using cell-free fetal
      DNA in spent culture medium will substitute for trophectoderm biopsy for heritable disorders
      screening and if it represents the total fetal genomic DNA.

      A total of 27 spent blastocyst media (SBM) from women undergoing IVF will be enrolled with
      agesless than44 years, whose indications for preimplantation genetic diagnosis for
      Euploid/Aneuploid embryo determination, were advanced maternal age, recurrent miscarriage, or
      recurrent implantation failure.We examined the presence of the cell free DNA in spent culture
      media and whether this DNA is reliable and can be representative of the chromosomal
      constitution of a blastocyst. So we can affirm that Noninvasive prenatal determination of
      fetal sex using cell-free fetal DNA (used here as a marker) provides an alternative to
      invasive techniques for some heritable disorders.
    

Detailed description: 
      Noninvasiveness is a common theme across medical specialties in the 21st century. In
      obstetrics, noninvasive prenatal testing provoked a paradigm shift in prenatal clinical
      practice through the sequencing of fetal cell-free DNA (cfDNA) in maternal plasma. However,
      current practice for preimplantation genetic testing for aneuploidy (PGT-A) requires
      trophectoderm biopsy, which entails technical challenges and embryo viability concerns. The
      possibility to overcome these issues reached the horizon in recent years, with the discovery
      that cfDNA released by the embryo to the culture media during the latest stages of
      development offers a new strategy to assess the preimplantation embryo genetically.

      Our study evaluates the possibility to isolate fetal cell-free DNA (cfDNA) from culture media
      and compare the results with TE biopsy samples from the same patients who underwent an IVF
      stimulation cycle with intracytoplasmic sperm injection at Al-Sharq Hospital between March
      2022 and April 2023. During the study's time frame, all embryos were cultured until the
      blastocyst stage. All enrolled patients underwent niPGD for gender determination using spent
      culture media as well as standard PGD with TE biopsy using Polymerase chain reaction
      technique for SRY gene. The embryos were individually cultured in 30-mL media droplets and
      embryo development progressed using sequential culture media, consistent with routine
      laboratory practice. For niPGD analysis, the blastocyst-stage culture media on Day 5 was
      collected (10-20 µl) immediately before TE biopsy was performed. Only embryos achieving a
      grade 1 (fragmentation less than 15%), were considered eligible for biopsy and vitrification.
      The embryo culture media samples underwent whole-genome amplification (WGA) using the
      DOPlify® WGA kit (PerkinElmer) to generate representative amplification of total DNA from
      spent culture media. The DOP-PCR-based WGA reproducibly amplifies total DNA to produce
      microgram quantities of amplified and purified genomic DNA in less than 3 hours. We aim to
      evaluate the concordance rates between trophectoderm biopsy results and cfDNA collected from
      spent media with an optimized protocol to obtain an adequate quantity and quality of genomic
      DNA. This interesting work is an important effort to establish the feasibility of the new
      noninvasive approach that can be a reliable alternative for chromosomal abnormalities
      assessment of in vitro cultured embryos which will lead to the birth of a living and healthy
      baby.

      Later on, we will perform the SRY gene detection for sex determination using the Polymerase
      Chain Reaction on the cell-free fetal DNA isolated from spent media samples and compare our
      results with the TB results.
    

Intervention name(s): Preimplantation Genetic Screening
Intervention type(s): Genetic
Intervention description(s): Non-invasive technique to investigate the presence of genetic disorders.
Other intervention name(s): PGS
Observational model: Other
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        The study samples will be collected from couples attending Orient Hospital (assisted
        reproduction and genetics) to carry out the in vitro fertilization process and would like
        to perform PGD for gender selection. After obtaining the approval of the ethical committee
        of Damascus University, they will be informed of the research objectives and sign the
        approve consent.
      
Biospecimen description: 
      Spent Culture Medium(SCM)
    
Keywords: Preimplantation Genetic Diagnosis, Spent Culture Medium, cell-free fetal DNA, trophectoderm biopsy