Intake of Vitamin E and C in Women of Reproductive Age: Results from the Latin American Study of Nutrition and Health (ELANS)

Vitamin E was identified as a lipophilic compound essential to maintain rat pregnancy. Low vitamin E intake during early pregnancy associates with congenital malformations and embryonic loss in animals and with miscarriage and intrauterine growth restriction in humans. Vitamin E protects cell membranes from lipoperoxidation and exerts non-antioxidant activities. Its function can be restored by vitamin C; thus, intake and circulating levels of both micronutrients are frequently analyzed together. Although substantial vitamin E inadequacy was reported worldwide, its consumption in Latin America (LatAm) is mostly unknown. Using data from the Latin American Study of Nutrition and Health (Estudio Latinoamericano de Nutrición y Salud, ELANS), we evaluated vitamin E and C intake in women of reproductive age (WRA) from eight LatAm countries and identified their main food sources. Two non-consecutive 24-h dietary recalls in 3704 women aged from 15 to 49 years and living in urban locations showed low average intake of vitamin E (7.9 mg/day vs. estimated average requirement (EAR) of 12 mg/day) and adequate overall vitamin C consumption (95.5 mg/day vs. EAR of 60 mg/day). The mean regional inadequacy was 89.6% for vitamin E and 36.3% for vitamin C. The primary food sources of vitamin E were fats and oils, as well as vegetables. Vitamin C intake was explained mainly by the consumption of fruit juices, fruits, and vegetables. Combined deficient intake of both vitamins was observed in 33.7% of LatAm women. Although the implications of low antioxidant vitamins' consumption in WRA are still unclear, the combined deficient intake of both vitamins observed in one-third of ELANS participants underscores the need for further research on this topic.Coca Cola Company///Estados UnidosHospital Infantil Sabará///BrazilInternational Life Science Institute//ILSI/ArgentinaUniversidad de Costa Rica//UCR/Costa RicaPontificia Universidad Católica de Chile///ChilePontificia Universidad Javeriana///ColombiaUniversidad Central de Venezuela//UCV/VenezuelaUniversidad San Francisco de Quito///EcuadorInstituto de Investigación Nutricional de Perú///PerúUCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

The effect of folic acid supplementation in the ovary and upon embryo development

Altered maternal nutrition around conception can affect oocyte and embryo development which influences later-life health outcomes. Low folate is related to poor reproductive outcomes. Folic acid (FA) supplementation and fortification have been effective strategies to avoid NTD. On the other hand, the FA levels in women of reproductive age are increased more than recommended, with unknown consequences. This project aims to determine the effect of high FA diet on the ovary and embryo development. C57BL/6 female mice at PND74 were fed with control (1mg FA/kg food) or high (5mg FA/kg food) FA diet for four weeks and culled at diestrus stage (PND102). In parallel, a group of animals were maintained on the control diet for another four weeks (PND130) or either mated and culled at 3.5 days post coitum (dpc). Ovaries and embryos were collected, RNA extracted and analysed by qPCR. Morphological and immunostaining analyses were also performed to determine the effect of FA in the ovary and blastocyst. High FA diet reduced expression of follicle developmental control genes at PND102 such as Fshr and Oct4, but also epigenetic writers like Ezh2 and Bmi1. In contrast, four weeks after FA diet release, the same genes were upregulated in the ovary. Females with a preconceptional high FA diet showed an increased mating period compared to the control group. The embryos of these mice showed reduced TE cells and lower expression of CDX2. In parallel, embryos exposed to high FA diet exclusively during preimplantation showed delayed development with decreased total cell number and lower expression of lineage markers (Oct4, NANOG and Gata6). High FA diet not only altered follicle growth factors during and after supplementation in a different pattern but also affected blastocyst biogenesis. This could impact on later-life health outcomes of the offspring

Folic acid induces intake-related changes in the mammary tissue transcriptome of C57BL/6 mice

Folic acid (FA) intake has been associated with increased breast cancer risk in some studies. Although underlying mechanisms are unknown, epigenetic modifications that persistently alter transcription have been suggested. We tested the hypothesis that high FA (HFA) intake alters the adult mammary transcriptome in a manner consistent with increased potential for carcinogenesis, detectable beyond the period of intake. C57BL/6 mice were fed control FA (CFA) (1 mg/kg diet) or HFA (5 mg/kg diet) diets for 4 weeks, followed by AIN93M maintenance diet for 4 weeks. Plasma 5-methyltetrahydrofolate, p-aminobenzoylglutamate and unmetabolised FA concentrations were greater (1.62, 1.56, 5.80-fold, respectively) in HFA compared to CFA mice. RNA sequencing of the mammary transcriptome (~20 million reads) showed 222 transcripts (191 upregulated) differentially expressed between groups. Gene Set Enrichment showed upregulated genes significantly enriched in Epithelial Mesenchymal Transition, Myogenesis and Apical Junction and downregulated genes in E2F targets, MYC targets and G2M checkpoint. Cancer was the most altered Disease and Disorder pathway, with Metastasis, Mammary Tumour and Growth of Tumour the most upregulated pathways. ChIP-seq enrichment analysis showed that targets of histone methyltransferase EZH2 were enriched in HFA mice. This study demonstrates HFA intake during adulthood induces mammary transcriptome changes, consistent with greater tumorigenic potential.</p

Sequence of primers for real time PCR.

<p>Rattus <i>HSD11B2</i> cDNA (GenBank acc. NM_017081), Rattus norvegicus DNA methyltransferase (cytosine-5) 1 (Dnmt1) cDNA (GenBank acc. no NM_053354).</p><p>Sequence of primers for real time PCR.</p

Methylation of <i>HSD11B2</i> in placentas attached to males in CpG sites of the promoter region (−378 to −275).

<p>(A). DNA of six placentas attached to males per group was extracted (each placenta came from different litter). Results are expressed as percentage of methylation (%) (B).</p

Expression of 11β-HSD2 in placentas attached to females and males.

<p><i>HSD11B2</i> mRNA was determined by real time PCR as described in Materials and Methods. Results represent the HSD11B2/β actin ratio of duplicate experimental determination of 6 different biological samples of placentas attached to females and males from different litters (A). Western blot analysis for 11β-HSD2 protein expression in placentas attached to males and females (B). 11β-HSD2 western blot signals were determined by densitometry and expressed as arbitrary units related to β-actin (C). *<i>p</i><0.05 compared to control; Mann-Whitney test.</p

Concentration of folates in placental tissue.

<p>Results are expressed as mean ± SD, (n = 6 placentas from different litters per group); P>0.05, Mann-Whitney test.</p><p>Concentration of folates in placental tissue.</p

Placental DNMT1expression (mRNA) related to β—Actin.

<p>DNMT1 mRNA was determined by real time PCR as described in Materials and Methods. Results represent the DNMT1/β actin ratio of duplicate experimental determination of 6 different biological samples of placentas attached to females and males from different litters; p>0.05, Mann-Whitney test.</p><p>Placental DNMT1expression (mRNA) related to β—Actin.</p

Methylation of <i>HSD11B2</i> in placentas attached to females in 5 CpG sites of the promoter region (−378 to −275).

<p>(A). DNA of six placentas attached to females per group was extracted (each placenta came from different litter). Results are expressed as percentage of methylation (%) (B). *<i>p</i><0.05; Chi-square test.</p

Maternal and fetal characteristics.

<p>Results are expressed as mean ± SD</p><p>^a: n = 6 pregnant rats per group</p><p>^b: n = 38</p><p>^c: n = 32</p><p>^d: n = 65</p><p>* <i>P</i><0.05, significantly different compared to controls, Student’s <i>t</i> test.</p><p>Maternal and fetal characteristics.</p