Effects of IVF culture conditions on the embryonic genome, methylome and transcriptome: a study to evaluate the safety and quality of different culture systems

The success rates of human assisted reproductive technologies (ART), such as in
vitro fertilisation (IVF), are relatively low. This is probably caused by a
combination of treatment and patient related factors, although the exact
aetiology and underlying mechanisms are still unknown. A growing body of
evidence shows that the IVF culture conditions, such as culture media or oxygen
tension, influence outcomes, including embryo development, pregnancy rates and
even short- and long-term health outcomes of the offspring. On the other hand,
a high rate of aneuploidy is seen in IVF embryos, which is also associated with
treatment failure. As it has been shown in cancerous cells, that global
hypomethylation is associated with chromosome instability (CIN), and that in
the post-zygotic stage of an embryo global demethylation takes place, that is
vulnerable for environmental cues, we hypothesize that IVF culture conditions
can alter the epigenome (in particular methylome) and transcriptome landscape
of preimplantation IVF embryos and, in turn, affect aneuploidy and treatment
success rates.

To study the effects of in vitro culture conditions, such as different culture
media or oxygen tension, on omic layers (i.e. genome, epigenome and
transcriptome) of preimplantation human embryos and to relate this to
chromosoom instability (CIN).

Observational, retrospective study using donated surplus cryopreserved human
embryos from ART treatments carried out in the past (between 2003-2006 and
2010-2013). Before cryopreservation on day 3, embryos were cultured in G3-1 or
G5-1 (Vitrolife), K-SICM (Cook), or HTF (Lonza) under either physiological (5%)
or atmospheric (20%) oxygen tension. After thawing, single cells will be
isolated either immediately, i.e. at cleavage-stage (day-3), or after extended
culture until the blastocyst-stage (day-5/6) in a time-lapse incubator. Single
cells will be analysed using single-cell multi-omic assays, e.g. single-cell
nucleosome, methylation and transcription sequencing (ScNMTseq), which
simultaneously profiles the chromatin accessibility, methylome and
transcriptome of a single cell. Parental genome data, obtained from DNA
isolated from saliva, will be used to distinguish between parental alleles in
the embryo. The latter is of great importance to investigate when and how
maternal molecular components, e.g. maternal RNA, are superseded by
embryonically derived components.

The embryo's will be cultured in the medium and oxygen level they were cultured
in before freezing. This can be one of the following media: HTF, G3, G5, Sydney
IVF Medium, and one of the following oxygne concentrations: 5% or 20%.

There is no risk or burden for participating couples, except for the donation
of their embryos and ceding a saliva sample.