Non-invasive preimplantation genetic testing (niPGT) - haplotyping by sequencing embryo spent culture medium

The rationale for this study is to develop a new methodology for
preimplantation genetic testing (PGT) that enables non-invasive PGT for
monogenic disorders (PGT-M) and structural rearrangements (PGT-SR) and enables
PGT for aneuploidy origin (PGT-AO) for embryo transfer ranking. We propose to
further develop our haplotyping-based PGT for cell-free DNA (cfDNA) from spent
culture medium (SCM) from an embryo. By developing this non-invasive PGT
(niPGT) method further, it has the potential to eventually replace the current
PGT methodology where an (invasive) embryo biopsy is needed. A non-WMO approval
by the METC aZM/UM (2020-1611) was previously obtained for reanalysis of only
the affected embryos (as inferred from conventional PGT) and for chromosomes
involved in the PGT indication. Here, we will further validate our genome-wide
haplotyping niPGT approach using SCM from both unaffected and affected embryos
and test for aneuploidy origin in the SCM and in biopsies from conventional PGT
cases.

Moreover, we will develop a method for PGT-AO in the SCM determining the rate
of meiotic/mitotic aneuploidies. Importantly, the proposed strategy is
non-invasive and makes use of data and materials (SCM and clinical data from
pregnancy) that are obtained in routine care. To develop and test the PGT-AO
approach, we will merely use embryos that have undergone PGT-M using the
haplotyping-based sequencing analysis or PGT-SR using the VeriSeq analysis.
Sequencing data is already generated in conventional PGT, but here only the
indication of interest is investigated. We will generate the aneuploidy origin
information from this sequencing data from TE biopsies obtained in conventional
PGT and from the sequencing data from the SCM. We will evaluate the predictive
value of the aneuploidy origin in TE and SCM for a healthy pregnancy and baby.
Based on the knowledge generated in this study, we could eventually create an
embryo transfer ranking system which could help to increase the success rate
per embryo transfer by first selecting the *best* embryo based on aneuploidy
origin.

1. Develop non-invasive PGT based on cfDNA in SCM (i.e. waste material) of IVF
embryos generated for PGT-M and PGT-SR.
2. Develop non-invasive PGT for aneuploidy origin (niPGT-AO) to assess the rate
of aneuploidies and the origin and assess the predictive value of aneuploidy
origin using pregnancy outcomes like implantation, miscarriage, live birth
rate.

This is an exploratory, diagnostic study that makes use of IVF waste material,
i.e. SCM samples, and IVF data of human preimplantation embryos (d3 and d5/6)
from couples that undergo an IVF/PGT treatment and clinical information about
resulting pregnancies. Following routine PGT, we will collect SCM samples and
store them at -20 ºC in the IVF laboratory, MUMC+. At the time of processing,
the SCM will be thawed and whole genome amplification (WGA) will be performed.
The amplified products will be whole genome sequenced and analyzed using our
haplotyping-based PGT approach. To this end, we will compare the genome of
preimplantation embryos that underwent conventional PGT with SCM-derived niPGT.
We will examine the genetic indication of interest and we will examine origin
of aneuploidies. Furthermore, epigenomic profiling will be performed to
allocate the origin of the cfDNA to either inner cell mass (ICM) or
trophectoderm (TE) and to account for maternal contamination. In addition, we
will link the epigenomic to genomic information of SCM of those embryos. The
data generated will be linked to clinical pregnancy outcomes, such as
implantation rate and miscarriage rate, live birth rate.

Participation in the study will not involve any risk or additional burden. No
additional visits, physical examinations, tests, blood samples other than
required for IVF/PGT are needed. There are no physical risks as the SCM will be
collected after conventional PGT biopsy procedures are performed and SCM is
considered as waste material. In this study we do not create or destroy any IVF
embryo other than for conventional PGT treatment.

With our niPGT approach, we will investigate whether the result for PGT-M or
PGT-SR in the SCM is concordant with conventional PGT. In a previous study, we
only investigated the affected embryos (non-WMO 2020-1611). Here, we will
investigate the SCM of both affected and unaffected embryos as has been
assessed by conventional PGT. Besides, we will also investigate the genome-wide
aneuploidy origin. Moreover, we will perform the genome-wide aneuploidy origin
as an extra chromosomal test on the TE biopsy compared to standard care. In
this study, we will develop PGT-AO that will elucidate the cellular origin of
aneuploidies and the segregational (meiotic/mitotic) origin. Since the
technique we will develop for non-invasive PGT is new and not yet
well-established, all samples and data will be provided with a non-traceable
code. Moreover, the results from the PGT-AO and niPGT will not be discussed
with the PGT couples. Moreover, the clinical significance of finding
mitotic/meiotic aneuploidies in TE biopsies as well as the representative value
of cfDNA in the SCM for the embryos* genetic constitution remains unclear. This
is to be explored and falls within the scope of this study.