Multi-center Study to Validate niPGT-A
Condition: Aneuploidy; Chromosome Abnormality; Infertility
MeSH terms: Infertility; Chromosome Disorders; Aneuploidy; Chromosome Aberrations
Study type: Observational

Summary: 
      Abnormal chromosome number, or aneuploidy, is common in human embryos. It is responsible for
      more than half of all miscarriages, and it is the leading cause of congenital birth defects.
      Besides, it has been described that aneuploidy may also affect embryo implantation.
      Therefore, selecting embryos that have the best chance of implanting and growing into a
      healthy baby is one of the most important steps in the field of assisted reproduction.

      Recent advances in genetic technologies, such as Next-Generation Sequencing (NGS), have
      allowed aneuploidy to be detected with greater sensitivity. The application of this technique
      to trophectoderm biopsies, taken from embryos before transfer to the uterus, has provided
      insight into the clinical impact of chromosomal status. This process of screening embryos to
      make sure they have the right number of chromosomes and to look for any structural
      abnormalities in the chromosomes is called Preimplantation Genetic Testing for Aneuploidy
      (PGT-A). It requires specific equipment and trained personnel that will add costs and risks,
      so non-invasive techniques are sought as an alternative. These non-invasive procedures have
      been explored by some groups analyzing the spent culture medium where the embryo is cultured
      up to the time of transfer or freezing. In daily routine, this media is discarded after
      finishing the embryo culture, but it has been reported that contains traces of embryonic
      cell-free DNA (cfDNA) that can represent the genetic load of the embryo. However, at the
      moment there is a high variability in results across studies, with a percentage of concordant
      results between the media and the trophectoderm biopsy ranging from 3.5 to 85.7%.

      Thus, the main objective of this project is to validate a new non-invasive method for PGT-A
      (niPGT-A), based on improved collection and analysis of the culture media to achieve higher
      rates of sensitivity and specificity and to decrease the effect of some intrinsic
      difficulties such as low embryonic cfDNA input, mosaicism and maternal contamination.
    

Detailed description: 
      Human embryos have higher aneuploidy rates (20-80%) than other species. A considerable
      proportion of these aneuploid embryos have the ability to reach the blastocyst stage.
      However, depending on the aneuploidy type, some will fail to implant in the uterus, while
      others will implant but will be unable to carry out early embryonic development
      (miscarriage), or very rarely, result in liveborn children with specific abnormalities. It is
      therefore important to identify aneuploid embryos. The identification of aneuploidies is
      especially important in embryos from patients with higher aneuploidy risk such as those with
      advanced maternal age (AMA), recurrent implantation failure (RIF), or recurrent miscarriage
      (RM).

      PGT-A technique analyse the full chromosome content of the embryo with high sensitivity and
      specificity but requires an invasive biopsy to obtain embryonic material for the genetic
      analysis. Thus, non-invasive methods to replace the existing invasive testing method would be
      useful in the improvement of maternal and fetal safety.

      Recently, there have been many research advances in the field of genetic testing. Cell-free
      DNA (cfDNA) has been observed in spent embryo culture media. The origin of the cfDNA at the
      blastocyst stage remains unknown and this has encouraged different research groups to carry
      out analysis of the spent culture media.

      Various studies were initially carried out to detect specific genes associated with monogenic
      disorders (MTHFR9, HBA1/HBA210, SRY11). Recently, non-invasive PGT-A has been developed, with
      highly variable results on the concordance rate (3.5%,59.1%, and 85.7%, 30.6%). The
      chromosomal status of the embryo from the DNA present in the spent culture medium was
      compared to the one obtained following the standard protocol using trophectoderm biopsy. The
      difference in the reported results can be related to the different methodologies applied
      because different amplification and detection methods -aCGH or NGS- were used. Moreover, the
      concordance rates were defined differently on each study, i.e. aneuploid results in spent
      culture media and trophectoderm biopsy could be considered concordant despite of showing not
      the same aneuploid chromosomes.

      The impact of culture conditions in the efficiency of the non-invasive approach has been
      thoroughly investigated. There could be influence of these relevant factors: external
      contamination from laboratory personnel or equipment, contamination with maternal DNA from
      granulosa cells (MCC) and mosaicism threshold for diagnosis..

      To improve the results of IVF (In vitro Fertilization) programs, there is a need to identify
      the embryo with highest implantation potential. Embryo chromosomal analysis allows the
      selection of euploid embryos, which have a higher implantation success rate.

      The development of a non-invasive PGT-A protocol will improve the current methodologies used
      to identify those euploid embryos avoiding the detrimental effect of the biopsy on the embryo
      and decreasing the economic cost.

      The main objective of the current study is to validate a new non-invasive method for PGT-A
      (niPGT-A), based on improved collection and analysis of the culture media to achieve higher
      rates of sensitivity and specificity and to decrease the effect of some intrinsic
      difficulties such as low embryonic cfDNA input and maternal contamination. The initial
      estimated sample size calculated was 3245embryos (each embryo is considered as a subject in
      the study), considering a dropout rate of 30%.

      Data exported from the medical records and source documents will be duly codified to protect
      the clinical and personal information of patients in accordance with the current legislation.
      This information will be exported to an electronic Case Report Form (eCRF). Data will be
      grouped and analyzed at Igenomix at three time points of the study: once 25 embryos of each
      center have been processed (to assess the implementation of the methodology), after the 30%
      of the samples have been processed (as an interim to check the results) and at the end of the
      study for the final analysis including the follow up of the clinical outcomes (defined
      following The International Glossary on Infertility and Fertility Care, 2017).

      After the interim analysis, the total sample size has been recalculated as 2620 samples,
      considering a drop-out rate of 5% according to the drop-out rate observed. Results of the
      interim analysis were published in Rubio et al., AJOG 2020.
    

Intervention name(s): PGT-A; niPGT-A
Intervention type(s): Diagnostic Test; Diagnostic Test
Intervention description(s): PGT-A will be carried out following the usual clinical practice: Trophectoderm biopsy samples from blastocysts are analyzed by NGS to screen for numerical chromosomal abnormalities.; Non-invasive preimplantation genetic test for embryo aneuploidy analyzing the spent culture media where the embryo is incubated up to the time of vitrification. This media contains traces of embryonic cell-free DNA (cfDNA) that can represent the genetic load of the embryo.
Observational model: Other
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Embryos from IVF patients between 20 and 44 years of age, undergoing PGT-A for any medical
        indication, with own oocytes or ovum donation cycles and with single embryo transfer (SET).
      
Biospecimen description: 
      -  Culture media: Embryonic cell-free DNA (cfDNA)

        -  Trophectoderm biopsy

        -  Inner cell mass biopsy
    
Keywords: Embryo; Blastocyst; Non-invasive; PGT-A; Chromosome; Aneuploidy; Spent culture medium; Trophectoderm biopsy