Higher Incidence of Embryonic Defects in Mouse Offspring Conceived with Assisted Reproduction from Fathers with Sperm Epimutations
Assisted reproductive technologies (ART) account for 1-6% of live births in developed countries. While most children conceived using ART are healthy, increases in birth and genomic imprinting defects have been reported; such abnormal outcomes have been attributed to underlying parental infertility and/or the ART used. Here, we assessed whether paternal genetic and lifestyle factors, that are associated with male infertility and affect the sperm epigenome, can influence ART outcomes. We examined how paternal factors, Dnmt3L haploinsufficiency and/or diet-induced obesity, in combination with ART (superovulation, in vitro fertilization, embryo culture and embryo transfer), could adversely influence embryo development and DNA methylation patterning in mice. While male mice fed high-fat diets (HFD) gained weight and showed perturbed metabolic health, their sperm DNA methylation was minimally affected by the diet. In contrast, Dnmt3L haploinsufficiency induced a marked loss of DNA methylation in sperm; notably, regions affected were associated with neurodevelopmental pathways and enriched in young retrotransposons, sequences that can have functional consequences in the next generation. Following ART, placental imprinted gene methylation and growth parameters were impacted by one or both paternal factors. For the embryos conceived by natural conception, the abnormality rates were similar for WT and Dnmt3L+/- fathers. In contrast, paternal Dnmt3L+/- genotype, as compared to WT fathers, resulted in a 3-fold increase in the incidence of morphological abnormalities in embryos generated by ART. Together, the results indicate that embryonic morphological and epigenetic defects associated with ART may be exacerbated in offspring conceived by fathers with sperm epimutations. Overall design: We examined the effect of Dnmt3L haploinsufficiency (Dnmt3L Het) and/or high fat diets (HFD) on the sperm epigenome (DNA methylation), as well as whether these two factors could influence embryo development after the use of assisted reproductive technologies (ART). Both wildtype (WT) and Dnmt3L Het male mice, starting at 8 weeks of age, were fed either a HFD with 60% kcal from fat or a matched-control-diet (MCD) with 10% kcal from fat; both diets contained 2 mg/kg folate. Males were fed the diets for 11-13 weeks, before breeding, ART or sperm collections. For natural matings, 2 CF1 females were placed into cages with one WT or Dnmt3L Het male; the day on which we observed a vaginal plug was denoted as E0.5. Midgestation embryos and placentas were collected 10 days later. For ART, eight week old CF1 females were superovulated by IP injection of 5 IU of PMSG followed by 5 IU HCG. IVF was performed in human tubal fluid using sperm from WT or Dnmt3L Het males on diets (MCD or HFD) and superovulated CF1 female oocytes. Fertilized oocytes were transferred to KSOM with 1/2 amino acids under mineral oil and cultured at 37°C, humidified and reduced oxygen conditions (5% O2, 5% CO2 and 90% N2) for 4 days. Blastocysts acquired from the embryo culture were transferred to 2.5 dpc pseudopregnant recipient CF1 female mice. Ten blastocysts were transferred to each female using a non-surgical embryo transfer device. Embryo transfer day is determined as embryonic day 3.5 (E3.5) and midgestation embryos and placentas were collected 8 days later.  Once breeding/ART was completed with the male mice, mature sperm from the cauda epididymides were collected with the swim-out method and kept at -80°C until DNA isolation was performed