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 births in developed countries. While most children conceived 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, haploinsufficiency for Dnmt3L, an important co-factor for DNA methylation reactions, 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 embryos conceived by natural conception, 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.

Capturing sex-specific and hypofertility-linked effects of assisted reproductive technologies on the cord blood DNA methylome.

BACKGROUND: Children conceived through assisted reproduction are at an increased risk for growth and genomic imprinting disorders, often linked to DNA methylation defects. It has been suggested that assisted reproductive technology (ART) and underlying parental infertility can induce epigenetic instability, specifically interfering with DNA methylation reprogramming events during germ cell and preimplantation development. To date, human studies exploring the association between ART and DNA methylation defects have reported inconsistent or inconclusive results, likely due to population heterogeneity and the use of technologies with limited coverage of the epigenome. In our study, we explored the epigenetic risk of ART by comprehensively profiling the DNA methylome of 73 human cord blood samples of singleton pregnancies (n = 36 control group, n = 37 ART/hypofertile group) from a human prospective longitudinal birth cohort, the 3D (Design, Develop, Discover) Study, using a high-resolution sequencing-based custom capture panel that examines over 2.4 million autosomal CpGs in the genome. RESULTS: We identified evidence of sex-specific effects of ART/hypofertility on cord blood DNA methylation patterns. Our genome-wide analyses identified ~ 46% more CpGs affected by ART/hypofertility in female than in male infant cord blood. We performed a detailed analysis of three imprinted genes which have been associated with altered DNA methylation following ART (KCNQ1OT1, H19/IGF2 and GNAS) and found that female infant cord blood was associated with DNA hypomethylation. When compared to less invasive procedures such as intrauterine insemination, more invasive ARTs (in vitro fertilization, intracytoplasmic sperm injection, embryo culture) resulted in more marked and distinct effects on the cord blood DNA methylome. In the in vitro group, we found a close to fourfold higher proportion of significantly enriched Gene Ontology terms involved in development than in the in vivo group. CONCLUSIONS: Our study highlights the ability of a sensitive, targeted, sequencing-based approach to uncover DNA methylation perturbations in cord blood associated with hypofertility and ART and influenced by offspring sex and ART technique invasiveness.

Protective and sex-specific effects of moderate dose folic acid supplementation on the placenta following assisted reproduction in mice.

Epigenetic defects induced by assisted reproductive technologies (ART) have been suggested as a potential mechanism contributing to suboptimal placentation. Here, we hypothesize that ART perturbs DNA methylation (DNAme) and gene expression during early placenta development, leading to abnormal placental phenotypes observed at term. Since folic acid (FA) plays a crucial role in epigenetic regulation, we propose that FA supplementation can rescue ART-induced placental defects. Female mice were placed on a control diet (CD), a moderate 4-fold (FAS4) or high dose 10-fold (FAS10) FA-supplemented diet prior to ART and compared to a natural mating group. ART resulted in 41 and 28 differentially expressed genes (DEGs) in E10.5 female and male placentas, respectively. Many DEGs were implicated in early placenta development and associated with DNAme changes; a number clustered at known imprinting control regions (ICR). In females, FAS4 partially corrected alterations in gene expression while FAS10 showed evidence of male-biased adverse effects. DNAme and gene expression for five genes involved in early placentation (Phlda2, EphB2, Igf2, Peg3, L3mbtl1) were followed up in placentas from normal as well as delayed and abnormal embryos. Phlda2 and Igf2 expression levels were lowest after ART in placentas of female delayed embryos. Moreover, ART concomitantly reduced DNAme at the Kcnq1ot1 ICR which regulates Phlda2 expression; FAS4 partially improved DNAme in a sex-specific manner. In conclusion, ART-associated placental DNAme and transcriptome alterations observed at mid-gestation are sex-specific; they may help explain adverse placental phenotypes detected at term and are partially corrected by maternal moderate dose FA supplementation.

Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model.

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 three 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 fertilisation, embryo culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. 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 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 (RRBS). 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 more pronounced in males. LARGE-SCALE DATA: The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143. LIMITATIONS REASONS FOR CAUTION: Although the combination of mouse ART utilised 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 foetal sex, are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, our findings support moderation in the dose of folic acid supplements taken during ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest.

Moderately Low Magnesium Intake Impairs Growth of Lean Body Mass in Obese-Prone and Obese-Resistant Rats Fed a High-Energy Diet.

The physical and biochemical changes resulting from moderately low magnesium (Mg) intake are not fully understood. Obesity and associated co-morbidities affect Mg metabolism and may exacerbate Mg deficiency and physiological effects. Male rats selectively bred for diet-induced obesity (OP, obese-prone) or resistance (OR, obese-resistant) were fed a high-fat, high-energy diet containing moderately low (LMg, 0.116 ± 0.001 g/kg) or normal (NMg, 0.516 ± 0.007 g/kg) Mg for 13 weeks. The growth, body composition, mineral homeostasis, bone development, and glucose metabolism of the rats were examined. OP and OR rats showed differences (p < 0.05) in many physical and biochemical measures regardless of diet. OP and OR rats fed the LMg diet had decreased body weight, lean body mass, decreased femoral size (width, weight, and volume), and serum Mg and potassium concentrations compared to rats fed the NMg diet. The LMg diet increased serum calcium (Ca) concentration in both rat strains with a concomitant decrease in serum parathyroid hormone concentration only in the OR strain. In the femur, Mg concentration was reduced, whereas concentrations of Ca and sodium were increased in both strains fed the LMg diet. Plasma glucose and insulin concentrations in an oral glucose tolerance test were similar in rats fed the LMg or NMg diets. These results show that a moderately low Mg diet impairs the growth of lean body mass and alters femoral geometry and mineral metabolism in OP and OR rats fed a high-energy diet.

Compromised oocyte quality and assisted reproduction contribute to sex-specific effects on offspring outcomes and epigenetic patterning.

Clinical studies have revealed an increased incidence of growth and genomic imprinting disorders in children conceived using assisted reproductive technologies (ARTs), and aberrant DNA methylation has been implicated. We propose that compromised oocyte quality associated with female infertility may make embryos more susceptible to the induction of epigenetic defects by ART. DNA methylation patterns in the preimplantation embryo are dependent on the oocyte-specific DNA methyltransferase 1o (DNMT1o), levels of which are decreased in mature oocytes of aging females. Here, we assessed the effects of maternal deficiency in DNMT1o (Dnmt1Δ1o/+) in combination with superovulation and embryo transfer on offspring DNA methylation and development. We demonstrated a significant increase in the rates of morphological abnormalities in offspring collected from Dnmt1Δ1o/+ females only when combined with ART. Together, maternal oocyte DNMT1o deficiency and ART resulted in an accentuation of placental imprinting defects and the induction of genome-wide DNA methylation alterations, which were exacerbated in the placenta compared to the embryo. Significant sex-specific trends were also apparent, with a preponderance of DNA hypomethylation in females. Among genic regions affected, a significant enrichment for neurodevelopmental pathways was observed. Taken together, our results demonstrate that oocyte DNMT1o-deficiency exacerbates genome-wide DNA methylation abnormalities induced by ART in a sex-specific manner and plays a role in mediating poor embryonic outcome.