Predictive biomarkers and initial analysis of maternal immune alterations in postpartum preeclampsia reveal an immune-driven pathology.

Introduction: Postpartum preeclampsia (PPPE) is an under-diagnosed condition, developing within 48 hours to 6 weeks following an uncomplicated pregnancy. The etiology of PPPE is still unknown, leaving patients vulnerable and making the identification and treatment of patients requiring postpartum care an unmet need. We aimed to understand the immune contribution to PPPE at the time of diagnosis, as well as uncover the predictive potential of perinatal biomarkers for the early postnatal identification of high-risk patients. Methods: Placentas were collected at delivery from uncomplicated pregnancies (CTL) and PPPE patients for immunohistochemistry analysis. In this initial study, blood samples in PPPE patients were collected at the time of PPPE diagnosis (48h-25 days postpartum; mean 7.4 days) and compared to CTL blood samples taken 24h after delivery. Single-cell transcriptomics, flow cytometry, intracellular cytokine staining, and the circulating levels of inflammatory mediators were evaluated in the blood. Results: Placental CD163+ cells and 1st trimester blood pressures can be valuable non-invasive and predictive biomarkers of PPPE with strong clinical application prospects. Furthermore, changes in immune cell populations, as well as cytokine production by CD14+, CD4+, and CD8+ cells, suggested a dampened response with an exhausted phenotype including decreased IL1ß, IL12, and IFNγ as well as elevated IL10. Discussion: Understanding maternal immune changes at the time of diagnosis and prenatally within the placenta in our sizable cohort will serve as groundwork for pre-clinical and clinical research, as well as guiding clinical practice for example in the development of immune-targeted therapies, and early postnatal identification of patients who would benefit from more thorough follow-ups and risk education in the weeks following an uncomplicated pregnancy.

Sex-based disparities in DNA methylation and gene expression in late-gestation mouse placentas.

BACKGROUND: The placenta is vital for fetal development and its contributions to various developmental issues, such as pregnancy complications, fetal growth restriction, and maternal exposure, have been extensively studied in mice. The placenta forms mainly from fetal tissue and therefore has the same biological sex as the fetus it supports. Extensive research has delved into the placenta's involvement in pregnancy complications and future offspring development, with a notable emphasis on exploring sex-specific disparities. However, despite these investigations, sex-based disparities in epigenetic (e.g., DNA methylation) and transcriptomic features of the late-gestation mouse placenta remain largely unknown. METHODS: We collected male and female mouse placentas at late gestation (E18.5, n = 3/sex) and performed next-generation sequencing to identify genome-wide sex differences in transcription and DNA methylation. RESULTS: Our comparison between male and female revealed 358 differentially expressed genes (DEGs) on autosomes, which were associated with signaling pathways involved in transmembrane transport and the responses to viruses and external stimuli. X chromosome DEGs (n = 39) were associated with different pathways, including those regulating chromatin modification and small GTPase-mediated signal transduction. Differentially methylated regions (DMRs) were more common on the X chromosomes (n = 3756) than on autosomes (n = 1705). Interestingly, while most X chromosome DMRs had higher DNA methylation levels in female placentas and tended to be included in CpG dinucleotide-rich regions, 73% of autosomal DMRs had higher methylation levels in male placentas and were distant from CpG-rich regions. Several DEGs were correlated with DMRs. A subset of the DMRs present in late-stage placentas were already established in mid-gestation (E10.5) placentas (n = 348 DMRs on X chromosome and 19 DMRs on autosomes), while others were acquired later in placental development. CONCLUSION: Our study provides comprehensive lists of DEGs and DMRs between male and female that collectively cause profound differences in the DNA methylation and gene expression profiles of late-gestation mouse placentas. Our results demonstrate the importance of incorporating sex-specific analyses into epigenetic and transcription studies to enhance the accuracy and comprehensiveness of their conclusions and help address the significant knowledge gap regarding how sex differences influence placental function.

The placenta is a crucial organ for a healthy pregnancy and proper fetal development, and its functions are often studied in mice. The placenta stems from the developing embryo, and therefore shares its sex. Male fetuses have higher risks of pregnancy complications and neurodevelopmental disorders, and these risks are linked to placenta functions. However, how the placenta's sex influences the proteins it contains­and therefore, how it helps the fetus develop­remains largely unknown. We used cutting-edge techniques to systematically examine late-pregnancy mouse placentas, cataloging the genes being expressed (i.e., sections of DNA used to make proteins) and the patterns of a specific DNA mark (called methylation) that controls gene expression. We identified several genes with important placental functions, such as protecting the fetus from viruses and responding to environmental changes, whose expression levels were sex-specific. We also observed differences in DNA methylation between male and female placentas. Most DNA methylation differences were on the X chromosomes, and the majority had higher methylation levels in female placentas. Conversely, on other chromosomes, most differences present an increased level of DNA methylation in male placentas. As methylation affects gene expression, we found links between the changes. Additionally, we found that some sex differences in the placenta were already present earlier in pregnancy. Our findings provide important insights into the molecular differences between male and female mouse placentas during late pregnancy. Including sex-specific analyses in placenta studies will improve our understanding of how the placenta ensures the healthy development of male and female fetuses.

An adult patient with Tatton-Brown-Rahman syndrome caused by a novel DNMT3A variant and axonal polyneuropathy.

Tatton-Brown-Rahman syndrome (TBRS) is a rare autosomal dominant overgrowth syndrome first reported in 2014 and caused by pathogenic variants in the DNA methyltransferase 3A (DNMT3A) gene. All individuals reported to date share a phenotype of somatic overgrowth, dysmorphic features, and intellectual disability. Peripheral neuropathy was not described in these cases. We report an adult patient with TBRS caused by a novel pathogenic DNMT3A variant (NM_175629.2: c.2036G>A, p.(Arg688His)) harboring an axonal length-dependent sensory-motor polyneuropathy. Extensive laboratory and molecular genetic work-up failed to identify alternative causes for this patient's neuropathy. We propose that axonal neuropathy may be a novel, age-dependent phenotypic feature in adults with TBRS and suggest that this syndrome should be considered in the differential diagnosis of patients with overgrowth, cognitive and psychiatric difficulties, and peripheral neuropathy.

O-GlcNAcylation: the sweet side of epigenetics.

Histones display a wide variety of post-translational modifications, including acetylation, methylation, and phosphorylation. These epigenetic modifications can influence chromatin structure and function without altering the DNA sequence. Histones can also undergo post-translational O-GlcNAcylation, a rather understudied modification that plays critical roles in almost all biological processes and is added and removed by O-linked N-acetylglucosamine transferase and O-GlcNAcase, respectively. This review provides a current overview of our knowledge of how O-GlcNAcylation impacts the histone code both directly and by regulating other chromatin modifying enzymes. This highlights the pivotal emerging role of O-GlcNAcylation as an essential epigenetic marker.

Repurposing disulfiram, an alcohol-abuse drug, in neuroblastoma causes KAT2A downregulation and in vivo activity with a water/oil emulsion.

Neuroblastoma, the most common type of pediatric extracranial solid tumor, causes 10% of childhood cancer deaths. Despite intensive multimodal treatment, the outcomes of high-risk neuroblastoma remain poor. We urgently need to develop new therapies with safe long-term toxicity profiles for rapid testing in clinical trials. Drug repurposing is a promising approach to meet these needs. Here, we investigated disulfiram, a safe and successful chronic alcoholism treatment with known anticancer and epigenetic effects. Disulfiram efficiently induced cell cycle arrest and decreased the viability of six human neuroblastoma cell lines at half-maximal inhibitory concentrations up to 20 times lower than its peak clinical plasma level in patients treated for chronic alcoholism. Disulfiram shifted neuroblastoma transcriptome, decreasing MYCN levels and activating neuronal differentiation. Consistently, disulfiram significantly reduced the protein level of lysine acetyltransferase 2A (KAT2A), drastically reducing acetylation of its target residues on histone H3. To investigate disulfiram's anticancer effects in an in vivo model of high-risk neuroblastoma, we developed a disulfiram-loaded emulsion to deliver the highly liposoluble drug. Treatment with the emulsion significantly delayed neuroblastoma progression in mice. These results identify KAT2A as a novel target of disulfiram, which directly impacts neuroblastoma epigenetics and is a promising candidate for repurposing to treat pediatric neuroblastoma.

Clearance of defective muscle stem cells by senolytics restores myogenesis in myotonic dystrophy type 1.

Muscle stem cells, the engine of muscle repair, are affected in myotonic dystrophy type 1 (DM1); however, the underlying molecular mechanism and the impact on the disease severity are still elusive. Here, we show using patients' samples that muscle stem cells/myoblasts exhibit signs of cellular senescence in vitro and in situ. Single cell RNAseq uncovers a subset of senescent myoblasts expressing high levels of genes related to the senescence-associated secretory phenotype (SASP). We show that the levels of interleukin-6, a prominent SASP cytokine, in the serum of DM1 patients correlate with muscle weakness and functional capacity limitations. Drug screening revealed that the senolytic BCL-XL inhibitor (A1155463) can specifically remove senescent DM1 myoblasts by inducing their apoptosis. Clearance of senescent cells reduced the expression of SASP, which rescued the proliferation and differentiation capacity of DM1 myoblasts in vitro and enhanced their engraftment following transplantation in vivo. Altogether, this study identifies the pathogenic mechanism associated with muscle stem cell defects in DM1 and opens a therapeutic avenue that targets these defective cells to restore myogenesis.

Mitigating the detrimental developmental impact of early fetal alcohol exposure using a maternal methyl donor-enriched diet.

Fetal alcohol exposure at any stage of pregnancy can lead to fetal alcohol spectrum disorder (FASD), a group of life-long conditions characterized by congenital malformations, as well as cognitive, behavioral, and emotional impairments. The teratogenic effects of alcohol have long been publicized; yet fetal alcohol exposure is one of the most common preventable causes of birth defects. Currently, alcohol abstinence during pregnancy is the best and only way to prevent FASD. However, alcohol consumption remains astoundingly prevalent among pregnant women; therefore, additional measures need to be made available to help protect the developing embryo before irreparable damage is done. Maternal nutritional interventions using methyl donors have been investigated as potential preventative measures to mitigate the adverse effects of fetal alcohol exposure. Here, we show that a single acute preimplantation (E2.5; 8-cell stage) fetal alcohol exposure (2 × 2.5 g/kg ethanol with a 2h interval) in mice leads to long-term FASD-like morphological phenotypes (e.g. growth restriction, brain malformations, skeletal delays) in late-gestation embryos (E18.5) and demonstrate that supplementing the maternal diet with a combination of four methyl donor nutrients, folic acid, choline, betaine, and vitamin B12, prior to conception and throughout gestation effectively reduces the incidence and severity of alcohol-induced morphological defects without altering DNA methylation status of imprinting control regions and regulation of associated imprinted genes. This study clearly supports that preimplantation embryos are vulnerable to the teratogenic effects of alcohol, emphasizes the dangers of maternal alcohol consumption during early gestation, and provides a potential proactive maternal nutritional intervention to minimize FASD progression, reinforcing the importance of adequate preconception and prenatal nutrition.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Early-Life Exposure to Environmental Contaminants Perturbs the Sperm Epigenome and Induces Negative Pregnancy Outcomes for Three Generations via the Paternal Lineage.

Due to the grasshopper effect, the Arctic food chain in Canada is contaminated with persistent organic pollutants (POPs) of industrial origin, including polychlorinated biphenyls and organochlorine pesticides. Exposure to POPs may be a contributor to the greater incidence of poor fetal growth, placental abnormalities, stillbirths, congenital defects and shortened lifespan in the Inuit population compared to non-Aboriginal Canadians. Although maternal exposure to POPs is well established to harm pregnancy outcomes, paternal transmission of the effects of POPs is a possibility that has not been well investigated. We used a rat model to test the hypothesis that exposure to POPs during gestation and suckling leads to developmental defects that are transmitted to subsequent generations via the male lineage. Indeed, developmental exposure to an environmentally relevant Arctic POPs mixture impaired sperm quality and pregnancy outcomes across two subsequent, unexposed generations and altered sperm DNA methylation, some of which are also observed for two additional generations. Genes corresponding to the altered sperm methylome correspond to health problems encountered in the Inuit population. These findings demonstrate that the paternal methylome is sensitive to the environment and that some perturbations persist for at least two subsequent generations. In conclusion, although many factors influence health, paternal exposure to contaminants plays a heretofore-underappreciated role with sperm DNA methylation contributing to the molecular underpinnings involved.

Pre-implantation alcohol exposure induces lasting sex-specific DNA methylation programming errors in the developing forebrain.

BACKGROUND: Prenatal alcohol exposure is recognized for altering DNA methylation profiles of brain cells during development, and to be part of the molecular basis underpinning Fetal Alcohol Spectrum Disorder (FASD) etiology. However, we have negligible information on the effects of alcohol exposure during pre-implantation, the early embryonic window marked with dynamic DNA methylation reprogramming, and on how this may rewire the brain developmental program. RESULTS: Using a pre-clinical in vivo mouse model, we show that a binge-like alcohol exposure during pre-implantation at the 8-cell stage leads to surge in morphological brain defects and adverse developmental outcomes during fetal life. Genome-wide DNA methylation analyses of fetal forebrains uncovered sex-specific alterations, including partial loss of DNA methylation maintenance at imprinting control regions, and abnormal de novo DNA methylation profiles in various biological pathways (e.g., neural/brain development). CONCLUSION: These findings support that alcohol-induced DNA methylation programming deviations during pre-implantation could contribute to the manifestation of neurodevelopmental phenotypes associated with FASD.

Alteration of the brain methylation landscape following postnatal inflammatory injury in rat pups.

Preterm infants are vulnerable to inflammation-induced white matter injury (WMI), which is associated with neurocognitive impairment and increased risk of neuropsychiatric diseases in adulthood. Epigenetic mechanisms, particularly DNA methylation, play a role in normal development and modulate the response to pathological challenges. Our aims were to determine how WMI triggered DNA methylation alterations in brains of neonatal rats and if such changes persisted over time. We used a robust model of WMI by injecting lipopolysaccharide (LPS) or sterile saline in the corpus callosum of 3-day-old (P3) rat pups. Brains were collected 24 hours (P4) and 21 days post-injection (P24). We extracted genomic DNA from the brain to establish genome-wide quantitative DNA methylation profiles using reduced representation bisulfite sequencing. Neonatal LPS exposure induced a persistent increased methylation of genes related to nervous system development and a reduced methylation of genes associated with inflammatory pathways. These findings suggest that early-life neuroinflammatory exposure impacts the cerebral methylation landscape with determining widespread epigenetic modifications especially in genes related to neurodevelopment.

Heart failure drug proscillaridin A targets MYC overexpressing leukemia through global loss of lysine acetylation.

BACKGROUND: Cardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation. METHODS: Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry. RESULTS: At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression. CONCLUSION: Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.

ATAT1 regulates forebrain development and stress-induced tubulin hyperacetylation.

α-Tubulin acetyltransferase 1 (ATAT1) catalyzes acetylation of α-tubulin at lysine 40 in various organisms ranging from Tetrahymena to humans. Despite the importance in mammals suggested by studies of cultured cells, the mouse Atat1 gene is non-essential for survival, raising an intriguing question about its real functions in vivo. To address this question, we systematically analyzed a mouse strain lacking the gene. The analyses revealed that starting at postnatal day 5, the mutant mice display enlarged lateral ventricles in the forebrain, resembling ventricular dilation in human patients with ventriculomegaly. In the mice, ventricular dilation is due to hypoplasia in the septum and striatum. Behavioral tests of the mice uncovered deficits in motor coordination. Birth-dating experiments revealed that neuronal migration to the mutant septum and striatum is impaired during brain development. In the mutant embryonic fibroblasts, we found mild defects in cell proliferation and primary cilium formation. Notably, in these cells, ATAT1 is indispensable for tubulin hyperacetylation in response to high salt, high glucose, and hydrogen peroxide-induced oxidative stress. We investigated the role of ATAT1 in the hematopoietic system using multicolor flow cytometry and found that this system remains normal in the mutant mice. Although tubulin acetylation was undetectable in a majority of mutant tissues, residual levels were detected in the heart, skeletal muscle, trachea, oviduct, thymus and spleen. This study thus not only establishes the importance of ATAT1 in regulating mouse forebrain development and governing tubulin hyperacetylation during stress responses, but also suggests the existence of an additional α-tubulin acetyltransferase.

Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-ß Signaling and Epigenomics.

BACKGROUND & AIMS: A generalized human pacemaking syndrome, chronic atrial and intestinal dysrhythmia (CAID) (OMIM 616201), is caused by a homozygous SGO1 mutation (K23E), leading to chronic intestinal pseudo-obstruction and arrhythmias. Because CAID patients do not show phenotypes consistent with perturbation of known roles of SGO1, we hypothesized that noncanonical roles of SGO1 drive the clinical manifestations observed. METHODS: To identify a molecular signature for CAID syndrome, we achieved unbiased screens in cell lines and gut tissues from CAID patients vs wild-type controls. We performed RNA sequencing along with stable isotope labeling with amino acids in cell culture. In addition, we determined the genome-wide DNA methylation and chromatin accessibility signatures using reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing. Functional studies included patch-clamp, quantitation of transforming growth factor-ß (TGF-ß) signaling, and immunohistochemistry in CAID patient gut biopsy specimens. RESULTS: Proteome and transcriptome studies converge on cell-cycle regulation, cardiac conduction, and smooth muscle regulation as drivers of CAID syndrome. Specifically, the inward rectifier current, an important regulator of cellular function, was disrupted. Immunohistochemistry confirmed overexpression of Budding Uninhibited By Benzimidazoles 1 (BUB1) in patients, implicating the TGF-ß pathway in CAID pathogenesis. Canonical TGF-ß signaling was up-regulated and uncoupled from noncanonical signaling in CAID patients. Reduced representative bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing experiments showed significant changes of chromatin states in CAID, pointing to epigenetic regulation as a possible pathologic mechanism. CONCLUSIONS: Our findings point to impaired inward rectifier potassium current, dysregulation of canonical TGF-ß signaling, and epigenetic regulation as potential drivers of intestinal and cardiac manifestations of CAID syndrome. Transcript profiling and genomics data are as follows: repository URL: https://www.ncbi.nlm.nih.gov/geo; SuperSeries GSE110612 was composed of the following subseries: GSE110309, GSE110576, and GSE110601.

Rapid Multiplexed Reduced Representation Bisulfite Sequencing Library Prep (rRRBS).

DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.

DNA methylation, environmental exposures and early embryo development.

The first crucial step in the developmental program occurs during pre-implantation, the time after the oocyte has been fertilized and before the embryo implants in the uterus. This period represents a vulnerable window as the epigenome undergoes dynamic changes in DNA methylation profiles. Alterations in the early embryonic reprogramming wave can impair DNA methylation patterns and induce permanent changes to the developmental program, leading to the onset of adverse health outcomes in offspring. Although there is an increasing body of evidence indicating that harmful exposures during pre-implantation embryo development can trigger lasting epigenetic alterations in offspring, the mechanisms are still not fully understood. Since physiological or pathological changes in DNA methylation can occur as a response to environmental cues, proper environmental milieu plays a critical role in the success of embryonic development. In this review, we depict the mechanisms behind the embryonic epigenetic reprogramming of DNA methylation and highlight how maternal environmental stressors (e.g., alcohol, heat stress, nutrient availability) during pre-implantation and assisted reproductive technology procedures affect development and DNA methylation marks.

Pre-implantation alcohol exposure and developmental programming of FASD: an epigenetic perspective.

Exposure to alcohol during in-utero development can permanently change the developmental programming of physiological responses, thereby increasing the risk of neurological illnesses during childhood and later adverse health outcomes associated with fetal alcohol spectrum disorder (FASD). There is an increasing body of evidence indicating that exposure to alcohol during gestation triggers lasting epigenetic alterations in offspring, long after the initial insult; together, these studies support the role of epigenetics in FASD etiology. However, we still have little information about how ethanol interferes with the fundamental epigenetic reprogramming wave (e.g., erasure and re-establishment of DNA methylation marks) that characterizes pre-implantation embryo development. This review examines key epigenetic processes that occur during pre-implantation development and especially focus on the current knowledge regarding how prenatal exposure to alcohol during this period could affect the developmental programming of the early stage pre-implantation embryo. We will also outline the current limitations of studies examining the in-vivo and in-vitro effects of alcohol exposure on embryos and underline the next critical steps to be taken if we want to better understand the implicated mechanisms to strengthen the translational potential for epigenetic markers for non-invasive early detection, and the treatment of newborns that have higher risk of developing FASD.