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

Telomere length is important for the maintaining the individual health of a species. However, recent studies have indicated that the telomere length of somatic cells can be drastically decreased in the offspring receiving in vitro fertilization (IVF) therapy, 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 NMN, a precursor of NAD+, during in vitro culture.While it could not be achieved in Sirt6 conditional knockout embryos. 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. Females were superovulated by an intraperitoneal injection of 10 IU pregnant mare serum gonadotrophin (PMSG), followed 48 h later by an intraperitoneal injection of 10 IU human chorionic gonadotrophin (hCG). Females were naturally mated with males after the hCG injection. All superovulated female mice were plugged vaginally after mating and randomly divided into the two groups (in vivo or in vitro). After in vivo development, D3.5 blastocysts (in vivo group) were collected by flushing each uterus of the plugged females with modified human tubal fluid media supplemented with 10% serum substitute supplement (mHTF-SSS). For the in vitro group, at 19-21 h post-hCG treatment, in vivo fertilized zygotes were collected from oviducts and cumulus cells were removed via digestion with hyaluronidase for 3-5 min. The zygotes were rinsed with mHTF-SSS, and placed in 20 ul drops of synthetic oviductal medium enriched with potassium (KSOM) covered with paraffin oil, and then equilibrated overnight in an incubator at 37℃ and 5% CO2. The zygotes were grown to the blastocyst stage at 37℃with 5% CO2 atmosphere in vitro. The blastocysts in both groups were chosen on the basis of their developmental status and morphology and quickly frozen in liquid nitrogen and stored at -80°C. RNA sequencing was performed on total RNA extracted from collected blastocysts in vivo and in vitro to identify genes that cause telomere shortening in vitro culture (In vivo: n=3; In vitro: n=3).