BACKGROUND:Vitamin B-12 is a cofactor in folate-mediated 1-carbon metabolism, which generates nucleotides {thymidylate [deoxythymidine monophosphate (dTMP)] and purines} and methionine. Depressed de novo thymidylate (dTMP) synthesis leads to uracil accumulation in DNA. OBJECTIVES:This study aimed to determine how B-12 availability affects mitochondrial DNA (mtDNA) integrity and mitochondrial function in skeletal muscle. B-12 deficiency was modeled in young-adult mice. Intramuscular B-12 injection in aged mice assessed the role of B-12 supplementation in age-related changes in skeletal muscle. METHODS:Male methionine synthase knockdown (Mtr+/-) and wild-type littermates (Mtr+/+) were weaned to either an AIN93G-based control diet containing 25 μg/kg vitamin B-12 (Mtr+/+, n = 8; Mtr+/-, n = 9) or a B-12-deficient (-B-12) diet containing 0 μg/kg vitamin B-12 (n = 9 per genotype) for 7 wk. Aged (20-22 mo) male C57BL/6N mice were acclimated to an AIN93G control diet 4 wk, then received either weekly injections of saline [vehicle control (30 μL 0.9% NaCl; n = 5) or B-12 (0.65 μg per 30 μL 0.9% NaCl; n = 6) in each of 2 hindleg muscles (1.25 μg B-12 total)] for 8 wk. Outcomes measured included maximal oxygen consumption rate, uracil in mtDNA (a biomarker of mtDNA integrity), mtDNA copy number, and mitochondrial mass. Data were analyzed using a 2-way analysis of variance in the Mtr+/- mouse model exposed to -B-12 diets and by a Student's t-test for B-12 supplementation in aged mice. RESULTS:The tibialis anterior (TA) muscle from Mtr+/- mice exhibited 50% lower (P = 0.01) maximal respiratory capacity of the electron transport chain than did TA from Mtr+/+ mice. Exposure to the -B-12 diet lowered the maximal capacity of complex I in mitochondrially rich muscle (soleus and mitochondria-rich portions of quadriceps and gastrocnemius) by 25% (P = 0.02). Uracil in mtDNA in red muscle and gastrocnemius was elevated ∼10 fold with exposure to -B-12 diet (P = 0.04 and P < 0.001, respectively). In aged mice, gastrocnemius complex IV activity was increased 2-fold with intramuscular B-12 supplementation (P = 0.04). CONCLUSIONS:Exposure to a-B-12 diet led to uracil accumulation in mtDNA and impaired maximal oxidative capacity in skeletal muscle. B-12 supplementation improved complex IV maximal capacity in gastrocnemius from aged mice, a model of age-related skeletal muscle decline.

Maternal nutrition plays a major role in neurodevelopment. This study hypothesized that maternal egg yolk supplementation will influence offspring choline concentrations, and these changes will be associated with resting-state network (RSN) activation and monoamine neurochemistry at weaning. Sows were fed a control (CON; n = 6) or an egg yolk-supplemented diet (EGG; n = 5) from gestation day 70 to weaning at postnatal day 21, and piglet liver and plasma were collected (CON, n = 24; EGG, n = 20) for choline and metabolite quantification at weaning. Maternal egg yolk supplementation increased liver choline and methionine concentrations in piglets (p < .05). Given our previous findings that egg yolk supplementation elevated executive control and cerebellar functional activation, the associations between metabolites and RSNs were examined. Liver choline was correlated with executive (r = 0.4584) and auditory network activation (r = -0.4751), while plasma choline was correlated with visual network activation (r = 0.5295) (p < .05). As we previously reported that maternal egg yolk supplementation altered piglet monoamine homeostasis, its links to choline and metabolite concentrations were assessed. Liver choline was associated with anterior hippocampal norepinephrine (r = 0.3275) and cerebellar serotonin metabolism (r = 0.3043) and liver TMAO was associated with anterior hippocampal norepinephrine (r = 0.3896), while plasma choline was linked to caudate serotonin (r = 0.3902) and dopamine (r = -0.3763) metabolism (p < .05). Overall, maternal egg yolk supplementation altered offspring choline and metabolite status, which was associated with RSNs and monoamines. These findings broaden our understanding of how one-carbon metabolite status may relate to neurochemical and functional brain outcomes in offspring.

This double-blind, randomized, controlled trial primarily aimed to determine how supplementation of a prenatal multivitamin with biotin levels above the Adequate Intake (AI) affected biotin status in mother-fetus dyads. Additionally, the correlation between biotin status and stress markers was also explored. Eighty healthy pregnant women were recruited between gestational week 12 and 16. They received a prenatal multivitamin containing either 150 μg (intervention) or 30 μg (control) biotin for 24 wk. Sixty-two participants completed the study. Fasting blood samples were collected at week 0, 12, and 24 and cord blood was collected at delivery to measure biotin status biomarkers and stress-related hormones. At both week 12 and 24, the intervention group had significantly higher serum biotin concentrations than the control group (p < 0.01). Cord blood biotin concentrations were also higher in the intervention than in the control group (p < 0.01). Maternal plasma concentrations of 3-hydroxyisovaleric acid-carnitine (3HIA-carnitine), a metabolite accumulated with marginal biotin insufficiency, were also more pronouncedly reduced in the intervention group (p = 0.035). Maternal stress hormone cortisol concentrations were lower (p = 0.04) in the intervention versus the control group and cortisol concentrations demonstrated a negative association with biotin concentrations in both maternal and cord blood (p < 0.05). Moreover, osteocalcin, a bone-derived hormone that suppresses chronic cortisol elevation, was positively associated with biotin concentrations in the cord blood (β = 0.013, p = 0.044). In conclusion, a higher-biotin prenatal multivitamin formulation with biotin content exceeding the AI led to increases in biotin biomarkers in the mother-fetus dyad. The higher concentrations of biotin status biomarkers were related to mitigation of stress indicators. Further research is needed to confirm the positive role of a higher biotin intake in improving maternal and fetal health. This trial was registered on ClinicalTrials.gov (NCT05673070).

A growing number of prenatal multivitamin (MVI) products use 5-methyltetrahydrofolate (5-MTHF) in place of the synthetic folic acid (FA), because FA needs to be reduced to 5-MTHF for use in cellular methylation reactions and excess FA consumption can increase unmetabolized FA (UMFA) concentrations in circulation. However, the efficacy of 5-MTHF supplementation on maintaining maternal and fetal folate status in comparison to FA has not been sufficiently investigated. This study examined whether using 6S-5-MTHF instead of FA in a prenatal MVI affected total folate and UMFA status in maternal and fetal tissues. Eighty first-trimester pregnant participants were randomized to receive either a prenatal MVI containing 1,000 μg DFE of 6S-5-MTHF (MTHF-MVI group) or 1,330 μg DFE of FA (FA-MVI group) for 24 weeks (n = 40/group) beginning at their second trimester. Fasting blood samples were collected at week 0, 12, and 24 of the study whereas cord blood and placental samples were collected at delivery. Thirty-one participants in each group completed the study. By week 24 of the study, 5-MTHF and total folate concentrations in maternal and cord serum and the placenta did not differ between groups. There were significantly fewer participants (7% vs. 31%) with detectable UMFA and lower average UMFA concentrations in maternal blood of the MTHF-MVI group vs. the FA-MVI group (p < 0.01). UMFA concentrations in the placenta were lower in the MTHF-MVI group than the FA-MVI group (p < 0.01). Few cord blood samples (n = 2) contained detectable UMFA and both were from the FA-MVI group. In conclusion, during the second and third trimesters of pregnancy, prenatal MVIs providing 6S-5-MTHF may be an efficacious alternative to FA to maintain total folate status while reducing UMFA concentrations in maternal and fetal compartments. More research is needed to delineate the influence of different folate forms on fetal health and developmental outcomes.Clinical trial registrationClinicalTrials.gov, identifier: NCT05673070.

BACKGROUND AND AIMS:Betaine, 20 g/day for 12 months, reduced liver injury in several trials in non-alcoholic steatohepatitis (NASH). Our aim was to determine the safety and efficacy of lower doses of betaine in clinically diagnosed metabolic dysfunction-associated steatotic liver disease (MASLD) and an elevated ALT. APPROACH AND RESULTS:We performed 3 pilot trials in participants with clinically diagnosed non-cirrhotic MASLD and ALT ≥50 U/L. In the first trial, 44 participants were randomized to 4 or 8 g daily for 12 weeks. In the second trial, 10 participants received 1 g/day for 24 weeks, while 16 participants received 2 g/day for 24 weeks in the third trial. The primary outcome was the percent decline in the abnormal component of ALT (ie, ALT >30 for males or >25 for females). Other outcomes included improvement in absolute ALT and AST, and other serologic tests of liver injury, including metabolomics-advanced steatohepatitis fibrosis (MASEF) score, cytokeratin 18, and pro-C3. Baseline and end-of-treatment data were compared with a paired t test. At baseline, more than 75% of participants had NASH when tested by the MASEF score. ALT, AST, cytokeratin 18, pro-C3, and MASEF score decreased significantly among participants receiving 8, 4​​​​​, and 2 g, but not 1 g. High-density lipoprotein increased in the 4 and 2 g cohorts; low-density lipoprotein did not change. Approximately 35% reported mild, transient gastrointestinal symptoms. CONCLUSIONS:Betaine 8, 4​​​​​​, and 2 g/day for 12-24 weeks significantly reduced ALT and other serologic markers of liver injury among participants with clinically defined MASLD and an elevated ALT.

In the present study, we aimed to investigate intratumoral karyotype diversity as well as the estrogen/progesterone effect on the cytogenetic profile of uterine leiomyomas (ULs). A total of 15 UL samples obtained from 15 patients were cultured in the media supplemented with estrogen and/or progesterone and without adding hormones. Conventional cytogenetic analysis of culture samples revealed clonal chromosomal abnormalities in 11 out of 15 ULs. Cytogenetic findings were presented by simple and complex chromosomal rearrangements (64% and 36% of cases, respectively) verified through FISH and aCGH. In most ULs with complex chromosomal rearrangements, the breakpoints did not feature clusterization on a single chromosome but were evenly distributed across rearranged chromosomes. The number of breakpoints showed a strong positive correlation with the number of rearranged chromosomes. Moreover, both abovementioned parameters were in a linear dependency from the number of karyotypically different clones per UL. This suggests that complex chromosomal rearrangements in ULs predominantly originate through sequential events rather than one hit. The results of UL cytogenetic analysis depended on the presence of estrogen and/or progesterone in the culture medium. The greatest variety of cytogenetically different cell clones was detected in the samples cultured without hormone supplementation. Their counterparts cultured with progesterone supplementation showed a sharp decrease in clone number, whereas such a decrease induced by estrogen or estrogen-progesterone supplementation was insignificant. These findings suggest that estrogen-progesterone balance is crucial for forming a UL cytogenetic profile, which, in turn, may underlie the unique response of the every karyotypically abnormal UL to medications.

Background/Objectives: The MTHFR677C>T gene variant results in a thermolabile MTHFR enzyme associated with elevated plasma homocysteine in TT individuals. Health risks associated with the TT genotype may be modified by dietary and supplemental folate intake. Supplementation with methyltetrahydrofolate (methylTHF) may be preferable to folic acid because it is the MTHFR product, and does not require reduction by DHFR to enter one-carbon folate metabolism. In the Mthfr677C>T mouse model for this variant, female 677TT (TT) mice have an increased incidence of hepatic steatosis. The objective of this study was to compare the effects of methylTHF and folic acid supplementation on hepatic steatosis and one-carbon metabolism in this model. Methods: Male and female C57BL/6J 677CC (CC) and TT mice were fed control (CD), 5xmethylTHF-supplemented (MFSD), or 5xfolic-acid-supplemented (FASD) diets for 4 months. Liver sections were assessed for steatosis by Oil Red O staining. One-carbon metabolites were measured in the liver and plasma. MTHFR protein expression was evaluated in the liver. Results: MFSD had no significant effect on plasma homocysteine, liver SAM/SAH ratios, or hepatic steatosis in males or females as compared to CD. MTHFR protein increased in MFSD TT female liver, but remained <50% of the CC. FASD had no effect on plasma homocysteine but it decreased the liver MTHFR protein and SAM/SAH ratios, and increased hepatic steatosis in CC females. Conclusions: MethylTHF and folic acid supplementation had limited benefits for TT mice, while folic acid supplementation had negative effects on CC females. Further investigation is required to determine if these effects are relevant in humans.

Background. Endometriosis is distinguished by its high prevalence and significant impact on the quality of life and reproductive health of women; however, its etiology and essential pathogenesis of remain uncertain so far. Modern research is increasingly focusing on immune, hormonal and genetic factors that share a common structure and participate in common metabolism — so-called single nucleotide polymorphisms (SNPs), including stress-induced phosphoprotein 1 (STIP1), which participates in tissue and cellular metabolism through transcription splicing and folding of RNA. The role of this protein, known as heat shock protein (HSP)-organizing protein, is being actively studied in cancer and hyperproliferative diseases. The role of the STIP1 gene and its product in the pathogenesis of adenomyosis appears to be studied insufficiently, thereby determining the relevance of the present study.Objectives. To evaluate the expression of stress-induced phosphoprotein 1 in eutopic endometrium and myometrium in women with isolated adenomyosis, as well as in combination with other benign hyperproliferative diseases of the reproductive system.Methods. Clinical study site: Clinical and Diagnostic Department of Ott Research Institute of Obstetrics, Gynecology and Reproductology. Design: an observational case-control study of patients with verified diagnoses of diffuse adenomyosis, uterine fibroids, and external genital endometriosis (main group — n = 55). The study group (n = 43) was divided into three subgroups: patients with isolated diffuse adenomyosis (AM, n = 16), adenomyosis in combination with uterine fibroids (AM + UF, n = 16), adenomyosis in combination with external genital endometriosis (AM + EGE, n = 11)), a comparison group — patients with uterine fibroids (n = 12) and a control group (n = 17) — women of reproductive age without gynecological diseases. The study was conducted from November 1, 2022 to September 30, 2023. The target indicator of the study was the level of relative mRNA (mRNA) expression of STIP1 gene (in RQ (Relative Quantity) units) in the uterus — adenomyosis glands, surrounding myometrium and endometrium. Histological evaluation of the endometrium served as an additional indicator. Statistical analysis of the results obtained, namely the relative level of mRNA expression, was carried out by the ΔΔСt method using the Expression Suit V1.0.3 program. (https://www.thermofisher.com/ru/ru/home/technical-resources/software-downloads/expressionsuite-software.html). The data analysis was performed using the GraphPad Prizm program (Insight Partners, USA). Differences between groups were evaluated by means of single factor ANOVA analysis (followed by post-hoc pairwise comparisons (Tukey test) of the values in each group. The differences were considered statistically significant at p < 0.05.Results. A high level of STIP1 gene expression was reported in myometrium of patients with isolated adenomyosis (more than 3-fold increase in relation to the comparison group — patients with uterine fibroids). In addition, myometrium of women with adenomyosis combined with uterine fibroids demonstrated a higher expression of STIP1 gene, compared to patients with isolated uterine fibroids (p < 0.01). The evaluation of the expression of mRNA of the STIP1 gene in the eutopic endometrium of patients with adenomyosis and women in the control group revealed no significant differences; however, STIP1 in the endometrium of women with adenomyosis was significantly lower than in the endometrium of both patients with uterine fibroids and women with adenomyosis combined with external genital endometriosis.Conclusion. Increased mRNA expression of STIP1 gene in myometrium in adenomyosis confirms its role in the pathogenesis of this disease. The role of the expression of the STIP1 gene and the corresponding protein is to be further clarified in order to assess its specificity and sensitivity as a diagnostic marker and to identify new approaches to the treatment of adenomyosis.

Background/Objectives: The folate Recommended Daily Allowance (RDA) for pregnant women is 600 μg/day dietary folate equivalents, which is equivalent to approximately 400 μg folic acid. Many prenatal supplements contain much higher doses of folic acid. The body’s ability to reduce synthetic folic acid to the metabolically active form may be exceeded with high levels of supplementation. The objective of this double-blinded randomized controlled intervention trial was to determine changes in unmetabolized folic acid and other biomarkers of folate and one-carbon metabolism in maternal and cord blood in response to a folic acid dose commonly found in prenatal supplements (800 μg/day) compared to the dose equivalent of the RDA (400 μg/day). Methods: Healthy pregnant women were randomized and provided supplements from their first prenatal visit (<12 weeks gestation) through delivery. Maternal blood was collected at baseline and delivery. Umbilical cord blood from the mothers was collected at delivery. Results: A repeated measures analysis of variance revealed that there was a significant group supplemental dose effect (p = 0.0225) on serum unmetabolized folic acid concentration in mothers but no difference in cord blood unmetabolized folic acid concentrations between groups. Mixed effects analysis found a significant overall effect of pre-pregnancy BMI (p = 0.0360) and length of previous folic acid supplementation (p = 0.0281) on serum folate concentrations. No treatment effect was seen in RBC folate concentrations. Choline concentrations were higher in cord blood from the 800 μg/day group compared to the 400 μg/day group, but there was no group effect in maternal choline concentrations. Conclusions: The results indicate that folic acid dose during pregnancy affects certain folate and one-carbon biomarkers, and these effects are not consistent between maternal and cord blood. Potential long-term effects of these results on both mothers and offspring are unknown and merit further investigation.

AimThe aim was to investigate mechanisms by which betaine improves hepatic insulin signaling in a dietary mouse model of insulin resistance and fatty liver.MethodsC57BL 6J mice were fed a standard diet (SF), a standard diet with betaine (SFB), a nutritionally complete high fat (HF) diet, or a high fat diet with betaine (HFB) for 14 weeks. In a separate experiment, mice were fed high fat diet for 18 weeks, half of whom received betaine for the final 4 weeks. Activation of insulin signaling in the liver was assessed by western blot. Insulin signaling was also assessed in insulin resistant primary human hepatocytes treated with betaine.ResultsAs compared with SF, mice receiving HF diet were heavier, had more hepatic steatosis, and abnormal glucose tolerance test (GTT). Betaine content in liver and serum was 50% lower in HF than in SF; betaine supplementation restored serum and liver betaine content. Betaine treatment of HF reduced whole body insulin resistance as measured by GTT. Betaine treatment of HF increased tyrosine phosphorylation of insulin receptor substrate-1 and phosphorylation (activation) of Akt, and increased hepatic glycogen content. In vitro, betaine reversed insulin resistance in primary human hepatocytes by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and of Akt.ConclusionBetaine supplementation reduced whole body insulin resistance and increased activation of insulin signaling pathways in the liver in a mouse model of insulin resistance and fatty liver created by feeding a nutritionally complete high fat diet for 14 weeks. Betaine also reduced liver injury as assessed by ALT and by liver histology. In vitro, betaine reversed insulin resistance by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and activation of downstream proteins in the insulin signaling cascade in insulin resistant primary human hepatocytes.

Prenatal multivitamins, including folic acid, are commonly consumed in excess, whereas choline, an essential nutrient and an important source of labile methyl groups, is underconsumed. Here, we characterized profiles of one-carbon metabolism and related pathways and patterns of DNA methylation in offspring exposed to excess or imbalanced micronutrients prenatally. Pregnant Wistar rats were fed either recommended 1× vitamins (RV), high 10× vitamins (HV), high 10× folic acid with recommended choline (HFolRC), or high 10× folic acid with no choline (HFolNC). Offspring were weaned to a high-fat diet for 12 weeks. Circulating metabolites were analyzed with a focus on the hypothalamus, an area known to be under epigenetic regulation. HV, HFolRC, and HFolNC males had higher body weight (BW) and lower plasma choline and methionine consistent with lower hypothalamic S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) and global DNA methylation compared with RV. HV and HFolNC females had higher BW and lower plasma 5-methyltetrahydrofolate and methionine consistent with lower hypothalamic global DNA methylation compared with RV. Plasma dimethylglycine (DMG) and methionine were higher as with hypothalamic SAM:SAH and global DNA methylation in HFolRC females without changes in BW compared with RV. Plasma trimethylamine and trimethylamine-N-oxide were higher in males but lower in females from HFolRC compared with RV. Network modeling revealed a link between the folate-dependent pathway and SAH, with most connections through DMG. Final BW was negatively correlated with choline, DMG, and global DNA methylation. In conclusion, prenatal intake of excess or imbalanced micronutrients induces distinct metabolic and epigenetic perturbations in offspring that reflect long-term nutritional programming of health.

Scope Disturbances in one‐carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr 677C >T variant on NAFLD. Methods and results This study uses the new Mthfr 677C >T mouse model for the human MTHFR 677C >T variant. Mthfr 677CC and Mthfr 677TT mice were fed control diet (CD) or folate‐deficient (FD) diets for 4 months. FD and Mthfr 677TT alter choline/methyl metabolites in liver and/or plasma (decreased S‐adenosylmethionine (SAM):S‐adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr 677TT , provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr 677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex‐based differences in methylation potential, betaine, sphingomyelin, and trimethylamine‐ N ‐oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr 677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. Conclusion This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr 677TT , whereas Mthfr 677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD‐related disorders.

BACKGROUND: The study of telomere length and influencing factors in early human development has both fundamental and applied importance. AIM: A comparative assessment of telomere length in the compartments of human blastocysts, and the analysis of the telomere length association with the quality of blastocysts, genetic imbalance and the maternal age. MATERIALS AND METHODS: The study was performed on trophectoderm and inner cell mass samples of 41 human blastocysts, 26 of which were genetically imbalanced according to preimplantation genetic testing and verification of its results. The microscope slides were prepared for further telomere detection in interphase nuclei by quantitative fluorescence in situ hybridization (Q-FISH). RESULTS: Telomeres in trophectoderm were longer than in inner cell mass, with their length varied from blastocyst to blastocyst. Telomere length in either trophectoderm or inner cell mass did not differ between genetically balanced and imbalanced blastocysts. There was a tendency towards a decrease in telomere length in the blastocyst compartments with increasing maternal age, however, a statistically significant correlation was not confirmed. The telomere length in the inner cell mass, but not in the trophectoderm, was associated with blasocysts’ quality based on the Gardner grade: medium quality blastocysts had longer telomeres than high quality blastocysts. CONCLUSIONS: Long telomeres in trophectoderm may be necessary for implantation and subsequent placentation. Telomere length can be considered among modifiers of the effects of karyotype abnormalities and other negative factors: the inheritance by an embryo of long telomeres apparently gives it a developmental advantage even when genetically imbalanced or has poor morphology. Implantation seems to be an important checkpoint for negative selection of embryos with “unsuccessful” combinations of telomere length, karyotype, and morphology.

BACKGROUND: Epigenetic genome reprogramming is an important determinant of human embryo development. However, its mechanisms remain poorly elucidated, especially in genetically unbalanced embryos. AIM: The aim of this study is the analysis of DNA methylation and hydroxymethylation levels in trophectoderm and inner cell mass of genetically balanced and unbalanced human blastocysts. MATERIALS AND METHODS: Twenty-two IVF-derived human blastocysts were enrolled in the study; of these blastocysts, 15 were genetically unbalanced and 7 — genetically balanced. Detection of 5-methylcytosine and 5-hydroxymethylcytosine was performed on trophectoderm and inner cell mass nuclei by indirect immunofluorescence. RESULTS: In genetically unbalanced blastocysts, the DNA methylation level was elevated in both compartments. The DNA hydroxymethylation level, in contrast, was elevated only in inner cell mass, whereas trophectoderm cells retained the same level as in genetically balanced embryos. These changes equalized the inner cell mass and trophectoderm DNA hydroxymethylation levels in genetically unbalanced blastocysts, while in genetically balanced ones the 5-hydroxymethylcytosine content in inner cell mass lagged behind that in trophectoderm. CONCLUSIONS: Genetic imbalance is associated with differential epigenetic changes in trophectoderm and inner cell mass cells of human blastocysts: DNA methylation level increases in both compartments while DNA hydroxymethylation level increases only in inner cell mass. The trophectoderm cells in genetically unbalanced blastocysts retain the same hydroxymethylation level as in genetically balanced ones, suggesting a possible explanation of the ability of karyotypically abnormal embryos to implant.

BACKGROUND:Folate and vitamin B12 (B12) are cofactors in folate-mediated 1-carbon metabolism (FOCM), a metabolic network that supports synthesis of nucleotides (including thymidylate [dTMP]) and methionine. FOCM impairments such as a deficiency or imbalance of cofactors can perturb dTMP synthesis, causing uracil misincorporation into DNA. OBJECTIVE:The purpose of this study was to determine how reduced expression of the B12-dependent enzyme methionine synthase (MTR) and excess dietary folic acid interact to affect folate distribution and markers of genome stability in mouse tissues. METHODS:Heterozygous Mtr knockout mice (Mtr+/-) model the FOCM-specific effects of B12 deficiency. Folate accumulation and vitamer distribution, genomic uracil concentrations, and phosphorylated histone H2AX (γH2AX) immunostaining were measured in male Mtr+/+ and Mtr+/- mice weaned to either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a high folic acid (HFA) diet (20 mg/kg folic acid) for 7 wk. RESULTS:Exposure to the HFA diet led to tissue-specific patterns of folate accumulation, with plasma, colon, kidney, and skeletal muscle exhibiting increased folate concentrations compared with control. Liver total folate did not differ. Although unmetabolized folic acid (UMFA) increased 10-fold in mouse plasma with HFA diet, UMFA accounted for <0.2% of total folate in liver and colon tissue. Exposure to HFA diet resulted in a shift in folate distribution in colon tissue with higher 5-methyl-THF and lower formyl-THF than in control mice. Mtr heterozygosity did not impact folate accumulation or distribution in any tissue. Mice on HFA diet exhibited higher uracil in genomic DNA and γH2AX foci in colon. Similar differences were not seen in liver. CONCLUSIONS:This study demonstrates that folic acid, even when consumed at high doses, does not meaningfully accumulate in mouse tissues, although high-dose folic acid shifts folate distribution and increases uracil accumulation in genomic DNA in colon tissue.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments—the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

It has been proven that the stress of the father can affect the phenotype of offspring, causing somatic, behavioral, hormonal and molecular changes. One of the hypothetical mechanisms responsible for the transmission of paternal effects to offspring may be a change in the spectrum of regulatory non-coding RNAs in spermatozoa. In this paper, we investigated the effect of paternal stress in models of post-traumatic stress disorder (PTSD) and depression on the representation of small RNAs (micro- and piwiRNAs) in the sperm of stressed animals. Male Wistar rats were subjected to stress in two paradigms (“stress–restress” and “learned helplessness”), which leads to the development of PTSD-like and depressive-like states in model animals, respectively. 48 days after the restress, sperm preparations were received and RNA was isolated. The spectrum of small RNAs was studied by NGS sequencing. In males with a PTSD-like condition, a change in the expression of 27 piwi RNAs and 77 microRNAs was detected compared with the control group. Among the targets of these miRNAs, it is possible to identify genes whose products may be involved in such mechanisms of transmission of paternal effects to offspring as changes in DNA methylation, histone modifications and RNA interference (Dnmt3a, Setd5, Hdac1, Mllt10, Mtdh), as well as genes associated with the functioning of insulin-like growth factor 2, the expression of which as previously shown, it is altered in the central nervous system in the offspring of males with a PTSD-like condition (Igf2, Igf2bp2, Igf2r). No changes in the representation of small RNAs were registered in males with a simulated depression-like state. The results indicate a pronounced effect of paternal stress on the spectrum of short non-coding RNAs in sperm cells in rats, however, it depends on the nature of the stress effect.

Maternal dietary levels of one-carbon (1C) metabolites (folic acid and choline) during pregnancy play a vital role in neurodevelopment. However, the impact of maternal dietary deficiencies on offspring stroke outcomes later in life remains undefined. The aim of this study was to investigate the role of maternal dietary deficiencies in folic acid and choline on ischemic stroke outcomes in middle-aged offspring. Female mice were maintained on either a control or deficient diet prior to and during pregnancy and lactation. At 10 months of age ischemic stroke was induced in male and female offspring. Stroke outcome was assessed by measuring motor function and brain tissue. There was no difference in offspring motor function; however, sex differences were present. In brain tissue, maternal dietary deficiency increased ischemic damage volume and offspring from deficient mothers had reduced neurodegeneration and neuroinflammation within the ischemic region. Furthermore, there were changes in plasma 1C metabolites as a result of maternal diet and sex. Our data indicate that maternal dietary deficiencies do not impact offspring behavior after ischemic stroke but do play a role in brain histology and one-carbon metabolite levels in plasma. Additionally, this study demonstrates that the sex of mice plays an important role in stroke outcomes.

ORs (95% CIs) of CRC by quartile of plasma DMG:choline ratio.