P-299 Influence of one carbon metabolism supplements over developmental dynamics and gene expression of genes related to DNA methylation machinery in mice
Authors: Reyes Palomares A , Dindini K , Rodriguez-Wallberg K 
Published: June 2023

Abstract

Study question
Does betaine and the active metabolite 5-MTHF influence cleavage dynamics and expression of genes related to methylation during preimplantation embryos?

Summary answer
Supplementation of betaine and 5-MTHF had no influence over morphokinetics of mice preimplantation embryos but induced transcriptional variations over methylation activity related-genes.

What is known already
After fertilization, the emerging genome is reprogrammed to enable the control of cellular differentiation all over downstream events and lead further developmental progression. One of the most determining processes defining these new genomic states in cell daughter is mediated by methylation. S-adenosylmethionine (SAM) is the main donor of methyl groups to DNA and its availability during highly active conditions can be sensitive to external conditions and dependent on one carbon metabolism (OCM). DNA methyltransferases (DNMT1, DNMT3a and DNTM3b) and methionine adenosyltransferase (MAT2a and MAT2b) are influenced by OCM and key components of DNA methylation machinery.

Study design, size, duration
This is a prospective experimental study conducted from November 2022 to January 2023. F1 hybrid (B6/CBA) fresh mice embryos were exposed to different culture conditions. A primary analysis consisted of the morphokinetics assessment of embryo development through time-lapse system up to expanded blastocyst (105 hours), including a score assessment based on blastocyst appearance. Subsequently functional analysis based on gene expression of selected genes was carried out over individual embryos.

Participants/materials, setting, methods
The zygotes were collected from the oviduct, washed and culture in KSOM supplemented with 5-MTHF and betaine at respectively concentrations of 50μM and 50μg/mL (THF/bet(50)), 10μM and 10μg/mL (THF/bet(10)) and no supplements for control. Geri incubator was used for culture at 37 °C, 6%CO2 and 5%O2. In a new assay, 4cells and 8cells embryos exposed to THF/bet(50) and control conditions were individually collected for pre-amplification and subsequent RT-qPCR quantification using GADPH as housekeeping gene.

Main results and the role of chance
A total of 54 mice embryos were used for the present study. The timings required for embryos to reach different developmental stages (t2, t4, t8 and tB) were manually selected and their comparison did not show any overall differences. A slightly faster trend could be observed for the THF/bet(50) group during the 3 first cleavage events (t2, t4 and t8), this group showed proportionally better morphology of blastocysts.
Expression analysis showed no variation for DNMT1; by contrast, greatest changes were observed for DNMT3a and DNMT3b whose relative expressions were downregulated at 4 cells ∼0.5 fold-changes (p &lt; 0.01) and highly increased for more than 2 fold-change in 8 cells in THF/bet(50) group. In addition, early downregulation at 4 cells was observed for either MAT2a and MAT2b with ∼0.5 fold-changes (p &lt; 0.01); however, at 8 cells levels were reestablished in both similar levels than control.
One carbon metabolism induced changes in gene expression of enzymes involved in de novo methylation of DNA DNMT3a and DNMT3a. In addition, Methyl adenyl transferases MAT2a and MAT2b were also influenced, particularly MAT2a which has a pivotal role relevant for genome activation in the embryo.

Limitations, reasons for caution
The translational approach is still limited due to known differences between kinetics of mice and human embryo. Intracellular concentration of folates and betaine in embryos are still unknown and the range of dosage used has been decided accordingly to previous studies showing a response in mice embryos under similar conditions.

Wider implications of the findings
Folate supplements are widely accepted for pregnant and women attempting to become pregnant, these are also recently incorporated in some IVF mediums. The influence of OCM in surrounding environment during highly active methylation activities of early embryo is still unknown, benefits or risks of these supplies require further assessment.

Trial registration number
not applicable