NAD+-dependent Sirt6 is a key regulator involved in telomere shortening of in vitro-cultured preimplantation embryos [Bisulfite-Seq]

Telomere length (TL) is important for the maintaining the individual health of a species. However, recent studies have indicated that blastocyst culture may increase the risk of telomere shortening in the offspring of those undergoing in vitro fertilization (IVF) treatment, however, the underlying molecular mechanism remains unknown. Sirt6 is a NAD+-dependent epigenetic regulator that has recently been found to play an important role in maintaining telomere stability. Here, we report for the first time that NAD+ levels are significantly lower in blastocysts cultured in vitro than that in blastocysts developed in vivo, leading to impaired Sirt6 function, further triggering telomere shortening of the inner cell mass and possibly affecting newborn offspring. This phenotype could be effectively mitigated by supplementation with nicotinamide mononucleotide (NMN), a precursor of NAD+, during in vitro culture, while it could not be achieved in Sirt6 conditional knockout embryos. Furthermore, mtROS accumulation and epigenetic modifications may also play an important role in this process. Our results reveal the mechanism by which in vitro culture induces telomere shortening in preimplantation embryos, providing a potential target for improving in vitro culture conditions. In the in vivo group, pseudopregnant female ICR mice (10-12 weeks old) were mated with vasectomized males 2.5 days prior to embryo transfer. In the in vitro group, female ICR mice (10 -12 weeks old) were employed as pseudopregnant moms and mated with vasectomized ICR males 3.5 days before embryo transfer. Surrogated ICR mice were selected based on the presence of a vaginal plug and then anesthetized. The uterine horn was removed through the back. Thirty blastocysts from the in vivo or in vitro group were transferred to a single recipient, respectively. The surrogate mother mice were housed in a clean cage with sufficient water and high‐fat chow for 19-21 days before their F1 pups were born. After birth, the tails of F1 pups were clipped at 1 week and 7 weeks of age respectively to extract genomic DNA for methylation sequencing (1W: In vivo: n=3, In vitro: n=3; 7W: In vivo: n=3, In vitro: n=3).