Noninvasive Chromosomal Screening for Embryo Selection Trial

Brief Title

Noninvasive Chromosomal Screening for Embryo Selection Trial

Official Title

Nonselection Pilot Study to Assess the Clinical Efficacy of Noninvasive Preimplantation Genetic Testing for Aneuploidy

Brief Summary

      To determine the diagnostic accuracy of non-invasive preimplantation genetic testing for
      aneuploidy (NI-PGT-A) for embryo selection.
    

Detailed Description

      Hypothesis:

      Analysis of cell free DNA (cfDNA) released from developing embryos into spent culture media
      (SCM) can serve as a non-invasive method for performing preimplantation genetic testing for
      aneuploidy (PGT-A). A non-selection study that compares clinical outcomes to the NI-PGT-A
      results of the transferred embryos in a blinded fashion will characterize the diagnostic
      accuracy by determination of positive and negative predictive values

      Justification:

      During the in vitro fertilization (IVF) process, the selection of a healthy embryo is limited
      by the inability of the microscopic appearance to reveal the chromosomal complement. Thus, in
      order to reliably select chromosomally normal embryos, preimplantation genetic testing for
      aneuploidy (PGT-A) is performed. PGT-A requires biopsy of the embryo, amplification of the
      DNA, then next generation sequencing (NGS) to determine chromosome copy number.

      While the clinical utility of PGT-A has been demonstrated, the process has limitations, the
      most significant of which is the requirement for embryo biopsy. Typically, 5-10 cells are
      removed from the TE after the zona pellucida has been breached. While the procedure can be
      safely and effectively performed by trained embryologists, the obvious concern for damage to
      the embryo is relevant. Even if implantation successfully occurs, concern has been raised
      that biopsy of the embryo at this stage can lead to complications such as miscarriage, or
      issues with placental function later in pregnancy. Furthermore, because the TE is destined to
      differentiate to placenta rather than the fetus itself, there is controversy regarding if the
      chromosomal analysis of these cells is always representative of the chromosomal complement of
      the fetus. For these reasons, and because TE biopsy is laborious and expensive, PGT-A has
      still not supplanted morphological evaluation as the clinical standard for embryo selection.

      An accurate noninvasive method for aneuploidy screening of embryos before implantation would
      be ideal. To this end, development of noninvasive PGT-A (NI-PGT-A) methods is an area of
      active investigation. The approach leverages the ability to isolate cell-free DNA (cfDNA)
      that is secreted from the blastocyst into the spent culture media (SCM). SCM is routinely
      discarded after the embryo has been removed from culture, however, minute amounts of DNA from
      the embryo can be isolated, amplified, then sequenced for chromosomal copy number analysis.
      The obvious advantage is that this process can occur without posing any harm to the embryo.
      An additional putative benefit of this noninvasive approach is that the DNA is secreted from
      both the TE and the inner cell mass (ICM), theoretically serving as a more accurate
      representation of the fetal karyotype than the DNA isolated from just the TE biopsy alone.

      Technical limitations of deploying NI-PGT-A are related to the limited quantity of DNA
      available in the SCM. Unlike other modes of genetic testing where ample amounts of genetic
      material is readily extracted from blood, amniotic fluid, or tissue biopsy, cell-free DNA
      secretion from blastocysts is exceptionally minute in quantity. However, laboratory protocols
      are developing rapidly, evidenced by numerous publications which have already demonstrated a
      high level of consistency when chromosomal copy number is analyzed by NI-PGT-A and
      traditional PGT-A in parallel.

      While these studies have illustrated proof of concept and preclinical validation, the true
      diagnostic accuracy can only be assessed by evaluating the predictive value of negative
      (euploid) and positive (aneuploid) results. Specifically, embryos found to be euploid should
      have a greater ability for sustained implantation that proceeds to live birth, while
      aneuploid embryos should have a dramatically lower rate of sustained implantation, or will be
      predictive of clinical aneuploidy. Without rigorous clinical validation, using these results
      for embryo selection could lead to misdiagnosis, causing selection of unhealthy embryos, or
      selection against embryos that could have been potentially viable.

      To determine if NI-PGT-A is clinically robust, we propose a non-selection, pilot, study,
      whereby embryo selection is performed by routine clinical methods (ie, morphological
      selection). However, after the clinical outcome has manifested, the SCM will be analyzed by a
      commercially available NI-PGT-A protocols in a blinded fashion. By correlating clinical
      outcomes to NI-PGT-A results, positive and negative predictive values can be calculated
      without bias. Because no intervention will be performed that deviates from the clinical
      routine, no additional risk will be posed to the embryo or parent.

      This study will serve as a pilot for a possible future trials. If reasonable predictive value
      of NI-PGT-A is demonstrated, a prospective, selection study could be justified to investigate
      the utility of NI-PGT-A in a clinical setting.

      Research Design:

      Patients will undergo IVF-ICSI per clinical indication, with plans to freeze all embryos for
      deferred embryo transfer. Embryo culture will follow routine protocols and embryo selection
      for transfer will be performed using standard morphological criteria. SCM from all
      blastocysts that appear viable will be collected and shipped to a reference lab (Sequence
      46). Specimens will be completely de-identified of patient information and labelled with an
      accession number. NI-PGT-A analysis will be performed using a commercial kit according to
      manufacturer's recommendations, with technicians blinded to clinical outcomes. When patients
      return for the embryo transfer cycle, routine clinical protocol will be followed for
      endometrial preparation. A single embryo will be selected according to routine morphological
      grading criteria, and embryo transfer will be performed with standard techniques. Initial
      pregnancy outcome will be documented with serum hCG testing. If hCG testing is positive,
      early pregnancy will be confirmed with ultrasound assessment per clinical routine. After all
      patients have completed treatment and clinical outcomes manifested (failed implantation,
      miscarriage, or ongoing pregnancy (defined as viability beyond 13 weeks), NI-PGT-A results
      will be released to the clinical team and correlated to clinical outcomes for calculation of
      predictive values. All on-going pregnancies will be offered routine prenatal screening for
      fetal aneuploidies.

      Additional details regarding laboratory protocols are as follows:

      Blastocyst culture Embryo culture will follow routine protocols. On day 3 post-fertilization,
      embryos will be repeatedly pipetted through a series of culture media droplets in order to
      lower potential for maternal cell contamination. Embryos will then be individually cultured
      in 35µL droplets of embryo culture medium to the blastocyst stage. On day 5 or day 6,
      blastocyst development and quality will be evaluated using standard morphological criteria.

      Sample collection and blastocyst vitrification Blastocyst culture medium (20µL) from selected
      embryos will be collected and transferred to PCR tubes and stored at -20°C until whole genome
      amplification (WGA). Blastocysts will be frozen via routine vitrification protocol and stored
      in liquid nitrogen per routine clinical protocol. Specimens will be completely de-identified
      of patient information and labelled with an accession number prior to shipping to reference
      lab (Sequence46).

      Whole genome amplification and DNA sequencing NI-PGT-A analysis will be performed upon
      receipt, using a commercial kit according to manufacturer's recommendations, with technicians
      blinded to clinical outcomes. DNA will be amplified by whole genome amplification (WGA) and
      barcoded to prepare libraries. The libraries are then loaded onto an Ion Chef instrument for
      template preparation by automated clonal amplification and then loaded onto a chip for DNA
      sequencing on an Ion Genestudio S5 Prime system, with approximately 150,000-200,000
      sequencing reads per sample. Data is analyzed using the Ion Reporter software (v5.10 or
      later). Ploidy will be measured using baseline, Ion ReproSeq Low-Coverage Whole-Genome (99M)
      Baseline. Standard quality control measures will be evaluated. Results will be released to
      the clinical team 6 weeks post-embryo transfer and correlated to clinical outcomes for
      calculation of predictive values.

      Patient population:

      Inclusion Criteria:

      Patient undergoing IVF-ICSI 21 - 35 years old at the time of enrollment Elective freeze-all
      cycle Planning for single embryo transfer

      Exclusion Criteria:

      Undergoing PGT-A, PGT-SR, PGT-M History of recurrent pregnancy loss History of recurrent
      implantation failure Planning on transfer of more than 1 embryo

      The justification for the age category is to control for age-related factors other than
      aneuploidy that may influence implantation, so variables that could compound predictive value
      calculations are minimized. Limiting participation to patients under 35 also optimizes embryo
      selection such that morphologically normal embryos are most likely healthy. Furthermore,
      patients in this age category have the lowest risk for clinical aneuploidies such as Down
      syndrome.

      Data management and storage:

      Research samples will be identified by a unique accession number, but no patient identifying
      information. The samples will be processed by Sequence46 with results associated with the
      accession number. The data will be uploaded via a secure cloud service, which will be
      accessed by the primary investigator to merge with the electronic case report form (eCRF)
      that will be stored locally at the Olive Fertility Centre on an encrypted hard drive that
      will be backed-up on a secondary local drive. The eCRF will contain the patients' unique
      medical record number and age, but will not document name, birthdate, or any other
      identifying information. Results of the NI-PGT-A analysis and clinical outcome will also be
      stored in the eCRF.

      Research data will be retained for 5 years after the date of publication.

      Statistical analysis:

      Diagnostic accuracy will be interpreted in the context of positive and negative predictive
      values.

      A positive result is a finding consistent with aneuploidy or mosaicism. A negative result is
      a finding consistent with euploidy.

      PPV = (Failed implantation + miscarriage + clinical aneuploidy)/ All Positive Results NPV =
      Ongoing pregnancy / All Negative Results

      Sample size calculation:

      One hundred-twenty (120) patients should suffice for this pilot study. Previous research has
      established that in patients aged 35 or younger, the ratio of euploid:aneuploid embryos is
      approximately 60:40. Of euploid embryos, approximately 70% will implant, of which, 85% should
      continue to viability. Of aneuploid embryos, approximately 25% will implant, of which >99%
      will miscarry; the risk of a clinical aneuploidy is less than 0.5% in women 35 and under.

      Given these probabilities, of the 120 patients to be enrolled, 72 euploid embryos are
      expected of which 50 should implant and 43 will continue as ongoing, viable pregnancies. Of
      the 48 that are expected to be aneuploid, 12 will implant, of which all should miscarry (the
      chance for a clinical aneuploidy in this population is less than 0.5%).

      Recruitment:

      Patients who are pursuing IVF-ICSI at the Olive Fertility Centre and meet inclusion/exclusion
      criteria will be recruited directly by the co-investigators. Flyers will be posted within the
      clinic.
    


Study Type

Observational


Primary Outcome

Predictive value of NI-PGT-A results with implantation rate.


Condition

Infertility

Intervention

No clinical intervention is planned; study is observational

Study Arms / Comparison Groups

 Study cohort
Description:  Single observational cohort of patients who meet study criteria

Publications

* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.

Recruitment Information


Recruitment Status

Other

Estimated Enrollment

120

Start Date

December 4, 2020

Completion Date

December 4, 2021

Primary Completion Date

December 4, 2021

Eligibility Criteria

        Inclusion Criteria:

        Patient undergoing IVF-ICSI at the Olive Fertility Centre

          -  21 - 35 years old at the time of enrolment

          -  Elective freeze-all cycle

          -  Planning for single embryo transfer

        Exclusion Criteria:

          -  Undergoing PGT-A, PGT-SR, PGT-M

          -  History of recurrent pregnancy loss

          -  History of recurrent implantation failure

          -  Planning on transfer of more than 1 embryo
      

Gender

All

Ages

21 Years - 35 Years

Accepts Healthy Volunteers

Accepts Healthy Volunteers

Contacts

Gary Nakhuda, MD, 604-816-5534, [email protected]

Location Countries

Canada

Location Countries

Canada

Administrative Informations


NCT ID

NCT04732013

Organization ID

NI-PGTA


Responsible Party

Principal Investigator

Study Sponsor

Olive Fertility Centre

Collaborators

 Thermo Fisher Scientific

Study Sponsor

Gary Nakhuda, MD, Principal Investigator, Olive Fertility Centre


Verification Date

January 2021