Project description:Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.

Project description:STUDY QUESTION: Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)? SUMMARY ANSWER: Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes. WHAT IS KNOWN ALREADY: Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear. STUDY DESIGN, SIZE, DURATION: Outbred female mice were fed 3 folic-acid supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilization, embryo culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Upon collection of midgestation embryos and placentas (n=74-99 embryos/group), all embryos were assessed for developmental delay and gross morphological abnormalities. Embryos and placentas were also examined at the epigenetic level. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1, and H19) in matched midgestation embryos and placentas (n=31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in midgestation placentas (n=6 normal placentas per sex/group) and embryos (n=6 normal female embryos/group; n=3 delayed female embryos/group) using reduced representation bisulfite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were far more pronounced in males. LIMITATIONS, REASONS FOR CAUTION: Although the combination of mouse ARTs utilized in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account fetal sex, are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our findings support moderation in the dose of folic acid supplements taken during ART.

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Effects of Assisted Reproductive Technology on Gene Expression in Heart and Spleen tissues of Adult Offspring

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