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.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

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.

Gender Disparities in Academic Outcomes Among Graduates of a Canadian MD-PhD Program.

PURPOSE: Women have traditionally been underrepresented in MD and MD-PhD training programs. Here, we describe the changing demographics of an MD-PhD Program over three distinct time intervals. METHODS: We designed a 64-question survey and sent it to 47 graduates of the McGill University MD-PhD program in Montréal, Québec, Canada, since its inception in 1985. We also sent a 23-question survey to the 24 students of the program in 2021. The surveys included questions related to demographics, physician-scientist training, research metrics, as well as academic and personal considerations. RESULTS: We collected responses from August 2020 to August 2021 and grouped them into three intervals based on respondent graduation year: 1995-2005 (n = 17), 2006-2020 (n = 23) and current students (n = 24). Total response rate was 90.1% (n = 64/71). We found that there are more women currently in the program compared to the 1995-2005 cohort (41.7% increase, p<0.01). In addition, women self-reported as physician-scientists less frequently than men and reported less protected research time. CONCLUSIONS: Overall, recent MD-PhD alumni represent a more diverse population compared with their earlier counterparts. Identifying barriers to training remains an important step in ensuring MD-PhD trainees become successful physician-scientists.

5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Impact of assisted reproduction, infertility, sex and paternal factors on the placental DNA methylome.

Children conceived using Assisted Reproductive Technologies (ART) have a higher incidence of growth and birth defects, attributable in part to epigenetic perturbations. Both ART and germline defects associated with parental infertility could interfere with epigenetic reprogramming events in germ cells or early embryos. Mouse models indicate that the placenta is more susceptible to the induction of epigenetic abnormalities than the embryo, and thus the placental methylome may provide a sensitive indicator of 'at risk' conceptuses. Our goal was to use genome-wide profiling to examine the extent of epigenetic abnormalities in matched placentas from an ART/infertility group and control singleton pregnancies (n = 44/group) from a human prospective longitudinal birth cohort, the Design, Develop, Discover (3D) Study. Principal component analysis revealed a group of ART outliers. The ART outlier group was enriched for females and a subset of placentas showing loss of methylation of several imprinted genes including GNAS, SGCE, KCNQT1OT1 and BLCAP/NNAT. Within the ART group, placentas from pregnancies conceived with in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) showed distinct epigenetic profiles as compared to those conceived with less invasive procedures (ovulation induction, intrauterine insemination). Male factor infertility and paternal age further differentiated the IVF/ICSI group, suggesting an interaction of infertility and techniques in perturbing the placental epigenome. Together, the results suggest that the human placenta is sensitive to the induction of epigenetic defects by ART and/or infertility, and we stress the importance of considering both sex and paternal factors and that some but not all ART conceptuses will be susceptible.

Testicular MTHFR deficiency may explain sperm DNA hypomethylation associated with high dose folic acid supplementation.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

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.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

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.

Developmental windows of susceptibility for epigenetic inheritance through the male germline.

Exposure of developing male germ cells to environmental insults has been linked to adverse effects in the offspring. One mechanism by which germ cell defects may be passed intergenerationally is through perturbations in the epigenome at the level(s) of DNA methylation, histone post-translational modifications and/or small non-coding RNAs. Epigenetic programs are particularly dynamic in germ cells undergoing erasure, re-establishment and maintenance of patterns, events potentially susceptible to prenatal and/or postnatal exposures. In this review, we focus on the epigenetic events occurring at each phase of male germ cell development including the prenatal period covering primordial germ cells and prospermatogonia and the postnatal period covering mitotic spermatogonia, meiotic spermatocytes and post-meiotic haploid spermatids and spermatozoa. Strong barriers to the passage of abnormal epigenetic patterns between generations are erected at two times of genome-wide epigenomic reprogramming, first in the germline in primordial germ cells and second, post-fertilization, during preimplantation development. Evidence from high resolution profiling studies that not all epigenetic marks are erased during germ cell and embryonic reprogramming provides a potential explanation for the intergenerational inheritance of abnormal epigenetic marks that may affect offspring health.

High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism.

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation.

STUDY QUESTION: Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? SUMMARY ANSWER: Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation. WHAT IS KNOWN ALREADY: There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities. STUDY DESIGN, SIZE, DURATION: Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex. MAIN RESULTS AND THE ROLE OF CHANCE: F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest.

Transient DNMT1 suppression reveals hidden heritable marks in the genome.

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.

The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105(+)) and myeloid (CD11b(+) and F4/80(+)) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis.

Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation.

3D Cohort Study: The Integrated Research Network in Perinatology of Quebec and Eastern Ontario.

BACKGROUND: The 3D Cohort Study (Design, Develop, Discover) was established to help bridge knowledge gaps about the links between various adverse exposures during pregnancy with birth outcomes and later health outcomes in children. METHODS: Pregnant women and their partners were recruited during the first trimester from nine sites in Quebec and followed along with their children through to 2 years of age. Questionnaires were administered during pregnancy and post-delivery to collect information on demographics, mental health and life style, medical history, psychosocial measures, diet, infant growth, and neurodevelopment. Information on the delivery and newborn outcomes were abstracted from medical charts. Biological specimens were collected from mothers during each trimester, fathers (once during the pregnancy), and infants (at delivery and 2 years of age) for storage in a biological specimen bank. RESULTS: Of the 9864 women screened, 6348 met the eligibility criteria and 2366 women participated in the study (37% of eligible women). Among women in the 3D cohort, 1721 of their partners (1704 biological fathers) agreed to participate (73%). Two thousand two hundred and nineteen participants had a live singleton birth (94%). Prenatal blood and urine samples as well as vaginal secretions were collected for ≥98% of participants, cord blood for 81% of livebirths, and placental tissue for 89% of livebirths. CONCLUSIONS: The 3D Cohort Study combines a rich bank of multiple biological specimens with extensive clinical, life style, and psychosocial data. This data set is a valuable resource for studying the developmental etiology of birth and early childhood neurodevelopmental outcomes.