HLTH 4401 Lecture 5 HS+JB collated handouts Fall 2024 PDF

Diet and developmental programming: the importance of B vitamins HLTH 4401 With Guest Lecturer: Hauna Sheyholislami Lecture Outline Part 1 Part 2...

Diet and developmental programming: the importance of B vitamins HLTH 4401 With Guest Lecturer: Hauna Sheyholislami Lecture Outline Part 1 Part 2 Part 3 The Context Neural tube Causes & development Consequences 1. Learning objectives 1. Folate/Folic acid 1. Folate transport/metabolism 2. Normal growth and 2. Neurulation dysfunction development 2. Pre-eclampsia 3. Pregnancy dietary 3. Placental dysfunction requirements 4. Paternal effects 5. Iron & LC-PUFA 1  Acknowledgements MacFarlane lab Nathalie Behan Danielle Leblanc Fernando Matias Myy Mikwar Carolyne Moussa Rachael Page Ian Zinck Collaborators Carole Yauk (HC) Francesco Marchetti (HC) Mike Wade (HC) Jacquetta Trasler (McGill) Lundi Ly Donovan Chan Funding: Health Canada, NSERC, CIHR Learning objectives Describe why micronutrients/minerals are critical for fetoplacental development, comparing and contrasting critical windows of exposure from both the mother/pregnancy and father/sperm perspectives Demonstrate, with evidence, how congenital anomalies can occur if specific micronutrients/minerals are in low supply 2  Learning objectives Demonstrate, with evidence, how specific (micro)nutrients/minerals can affect placental development, and explain what such scenarios mean for pregnancy and offspring outcomes both in the short and long-term Learning objectives Demonstrate, with evidence, how micronutrients/minerals can programme offspring outcomes through the paternal lineage, and explain what such scenarios mean for the offspring both in the short and long-term 3  Learning objectives Demonstrate, with evidence, how micronutrients/minerals may alter epigenetic pathways, identify at which sites these changes may occur, and hypothesise what such changes may mean for the pregnancy/offspring Hypothesise ways to mitigate micronutrient- mediated pregnancy complications and offspring maldevelopment 4  Normal Growth and Development Timing and characteristics of organ development are coordinated with the events of birth and weaning to ensure survival of the offspring Developmental Milestones movements / in blastel morphogenetic by Gastrulation -characterized – Formation of 3 germ layers (ectoderm, mesoderm, endoderm), gut, notochord by cell migration/division – Primitive body plan established Organogenesis – Formation of organs/organ systems Gastrulation Morphogenesis llul Ce erent ar ation i dif f – Development/differentiation of structures and form Cellular differentiation – Specialisation of cells Functional maturation Fu m nc at tio ur na at l – Attainment of functional capacity of cell, tissue, organ io n 5  Pregnancy is a time of increased nutrient requirements Non-pregnant Lactation Lactation 1st trimester 2nd trimester 3rd trimester (~19-50 years) 0-6 mo PP 6-12 mo PP Energy (kcal/day; 2.4 0 340 452 330 400 above non-pregnant) Folate (μg/day) 400 600 500 Vit B12 (μg/day) 2.6 2.8 Iron (mg/day) 18 27 9 Calcium (mg/day) 1000 1000 1000 α-linoleic acid (n-3 1.1 1.4 1.3 PUFA) (g/day) Linoleic acid (n-6 11 13 13 PUFA) (g/day) Fibre (g/day) 25 28 29 Health Canada Institute of Medicine Folate / Folic Acid I not. form Bioactive form is tetrahydrofolate (THF) Necessary for methyl transfer reactions, which are essential for purine and pyrimidine synthesis = required for DNA synthesis! Rapidly dividing cells are often folate dependent C lack of folate > cells will - stop proliferating Rev Med Hosp Gen Mex (2015) 78:135-143 6  Folate in pregnancy In pregnancy, there are some tissues that are dividing very rapidly ↳ placenta a embryo Greenberg (2011) Rev Obstet Gynecol 4:52-59 Fekete (2012) Nutr J 11:75 head - Developmental cranial neuropore 21-23 weels , Milestones 1 closure at ~25th day spiral cord zips up I head brain spinal cord will form along here closure at 2 ~27th day caudal neuropore word http://php.med.unsw.edu.au/embryology/index.php?title=File:Stage11_sem13c.jpg Freepik via Flaticon & base of spinal 7  ↳ Notochord - > signal overlying ectoderm to dev - > neural plate > - folds - > neural take , as platefolds -> neural drive PNS (ganglion , Schwa derive crests pinch off melanocytes ↳ CNs (Grain+ spinal cord) How do we get there? Neurulation neural groove neuroectoderm neural crest median hinge point https://basicmedicalkey.com/establishment-of-the-basic-embryonic-body-plan/ neural plate neural crest ectoderm Position of the neural plate in mesoderm endoderm relation to the germ layers neural crest Folding of the neural plate to paraxial mesoderm neural groove form the neural groove neural tube Dorsal closure of the neural folds to for neural tube and neural crest neural tube Maturation of the neural axial mesodermal somite tube and its position relative structure , notochord endoderm to the other structures notochords & somites G Schoenwolf via http://ftp.columbia.edu/itc/hs/medical/humandev/2004/Chapt4-Ectoderm.pdf 4-2. The neural plate folds in stages to form the neural tube. (Scanning electron micrographs of chick embryos provided by choenwolf.) A. Position of the neural plate in relation to the nonneural ectoderm, the mesoderm, and the endoderm. olding of the neural plate to form the neural groove. C. Dorsal closure of the neural folds to form the neural tube and neural. D. Maturation of the neural tube and its position relative to the axial mesodermal structure, notochord, and somites (derived m the paraxial mesoderm). (Adapted from Jessell & Sanes, Principles of Neuroscience 4th edition, 2002, E. Kandel editor) Neural induction-formation of the neural plate ural induction is the first step whereby the uncommitted or naïve ectoderm becomes committed to the 8 ral lineage. During gastrulation, signals from the node or its derivative, the notochord, induce mmitment. Classical studies led to the notion that inducing substances, secreted by the underlying  Neurulation step by step O https://youtu.be/lGLexQR9xGs ↳ neural tube x close properly > - consequences 9  Neural take defects severe congenital defects - Regions of neural tube closure proposed based on genetic evidence Anencephaly: failure of neural plate fusion in region 2 Fig. 4-6. Neurulation in the human embryo. (A)Dorsal and transverse sections of a 22 day human embryo initiating neurulation. Both anterior and posterior neuropores are open to the amniotic fluid. (B)Dorsal view of the neurulating human embryo a day Spina bifida: failure of posterior neuropore to close later.The anterior neuropore region is closing while the posterior neuropore remains open.(C)Regions of neural tube closure postulated by genetic evidence (superimposed on newborn body). (D) Anencephaly is caused by the failure of neural plate fusion (failure of region 5 to fuse) in region 2. (E) Spina bifida is caused by the failure of region 5 to fuse (or of the posterior neuropore to close).(C-E after Van Allen et al. 1993.)(Gilbert, Developmental Biology, 6th edition) Van Allen (1993) Gilbert Developmental Biology, 6th Edition II. Secondary neurulation: Most common birth defect Caudal to the posterior neuropore, the neural tube is formed by the process of secondary neurulation (Figure 4-7). A rod like condensation of mesenchymal cells forms beneath the dorsal ectoderm↳ ofnuval tail bud. Within the mesenchymal rod, a central canal forms by cavitation. This central canal becomes ~ the table zip up x O all the way Chole a posterior neural continuous with the one formed during primary neurulation and closure of the posterior neuropore. pore) Because of the diminished development of the tail bud in humans, secondary neurulation is not a ) ↳ cord dev spinal prominent process. outside body & babes are viable but comorbidities : paralysis below opening that occurs motor impairment , / sexual dysfunct. bladder dysfunction More severe neural tube defects Fig. 4-7.Secondary neurulation in the caudal region of a 25 -somite chick embryo. (From Catala et al. 1995; photographs -> nonviable courtesy of N.M. Le Douarin.)(Gilbert, Developmental Biology, 6th edition) anacephaly III. The neural tube forms the primordia of the central nervous system: Even before the neuropores have closed the future brain and spinal cord are recognizable and the brain becomes subdivided into a forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon) (Figure 4-8, 4-9). The increased volume of the early brain is the result of an increase in cavity size, not tissue growth. In the chick embryo, brain volume expands 30 fold between Via A MacFarlane YouTube Video: https://youtu.be/OpTX8WZbtvc 4-8 10 Nature? Nurture? Resilience? Vulnerability? Exposure to (stress) hormones Nutrition (Epi)Genetics Microbiome Inflammation Oxidative stress Pre-/peri-conception conditions Postnatal environments Risk factors Low circulating folate/folic acid levels (possibly other 1-carbon factors like B12, B6, myo-inositol, choline and methionine) contribute to pathogenesis of mural tube defects Previous pregnancy where fetus had an NTD Close relative born with an NTD Diabetes Obesity Medication use (e.g. sodium valproate or valproic acid) starzor - Ray (2007) Epidemiology 18, 362-366 Petersen (2019) Am J Epidemiol 188, 1136-1143 11  Folic acid supplementation during periconceptional 3 months period (first leading Prevents ~70% of neural tube defectsap to preg + 11 after preg) Observational studies 60s-70s ↳ women less w/higher likely fold to have estat babies us folic acid NTD ↑ given to women previously have Gaby u/ NTD to prevent2nd baby u/ NTD ↳ 70% reduction observed days http://php.med.unsw.edu.au/embryology/index.php?title=File:Stage11_sem13c.jpg (2016) SUNY Downstate Medical Center, Medical Research Library at Brooklyn via https://www.dentalcare.com/en-us/professional-education/ce-courses/ce311/levels-of-evidence 12  But the stork reality is… ~50% of pregnancies in Canada are unplanned Why would a women not planning to become pregnant take a prenatal folic acid supplement? https://moderndaymidwives.files.wordpress.com/2013/05/stork.jpg Mandatory folic acid fortification In 1998, mandatory fortification of white flour = - 8 and “enriched” cornmeal and pasta ⑦ foliz acido Increased daily folic acid intake by ~150 ug other Vitamins But did this do anything to prevent NTDs? 13  Public health success! ~45% reduction in NTD prevalence! NEJM (2007) 357:2 UK has highest NTD Rates in Europe , fortification policy is still under revision LX mandated as of 2019) https://www.theguardian.com/society/2018/oct/14/folic-acid-to-be-added-to-flour-in-effort-to-reduce-serious-birth-defects https://medicalxpress.com/news/2020-01-uk-folic-acid-fortification-foods.html 14 https://www.gov.uk/government/consultations/adding-folic-acid-to-flour/proposal-to-add-folic-acid-to-flour-consultation-document - Mandatory fortification of flour with folic acid has been considered by the UK government on several occasions and always deferred. The efficacy of fortification has not been in question and detailed consideration regarding safety has been reassuring Mandatory fortification of folic acid provides a "public health safety net" and could potentially prevent about half of all cases of neural tube defects https://medicalxpress.com/news/2020-01-uk-folic-acid-fortification-foods.html Wald (2019) Archives of Disease in ChildhoodDOI: 10.1136/archdischild-2019-318534 15  Diet & the gut microbiome Diet influences gut microbial composition Alterations of gut microbiome can affect: driven by dets – Immune responses – Intestinal homeostasis – Metabolic pathways “Western diets” containing less fibre/vegetables… – Loss of important microbial species in gut Mediterranean diet – healthy standard – Anti-inflammatory properties – Higher concentrations of “healthy” bacteria, lower concentrations of pathogenic bacteria Rajoka (2017) Food Sci Human Well 6(3):121-130. Mechanism: could the gut microbiome play a role? in vitamin BR2 & folate level ? Bacterial species in the gut possess the biosynthesis pathways to produce folate and vitamin B12 Lactobacillus plantarum, L. sakei, L. delbruekcii, L. reuteri, L. helveticus, L. fermentum Bifidobacteria 2 able alter [micronutrient] a may be -s availability during pregnancy Phys.org 16  Mechanism: could the gut microbiome play a role? - In influencingfetal health Outco Bacterial species in the gut possess the biosynthesis pathways to produce purines and pyrimidines L. plantarum, B. longum infantis Reduce apoptosis, increase cell proliferation = optimal embryogenesis Phys.org in pregnancy of fortification Other benefits? & exposure w/ respect to placental fur when men consume folic - acid Menton (2007) https://answersingenesis.org/human-body/the-placenta/ Freepik Flaticon 17  Folate & the placenta Maltep J Clin Invest (2010) 120:1016–1025 High rates of: Proliferation Apoptosis Restructuring of ~ vasculature a placental struc Cellular differentiation Via A MacFarlane 18 Evidence : Folate > - imp for placental genesis Folate transporters shuttle fetus ↳ exist > - -> of gene expression for folate metabolism proteins ↳ placenta has high l 1 ↳ receptors reduced folate carriers active , , transporters Folate transport & metabolism Solanky (2010) 31:134-143 Dynamic expression of folate-dependent enzymes in the placenta suggests active metabolism Placental Requirements for folate changes I all prolife differ Overtime : growth remod , , Methionine Methylene- Cystathionine synthase tetrahydro- beta folate synthase First trimester Third trimester reductase Solanky (2010) 31:134-143 19  Why folute support DNA synthesis o cell prolif Folate-dependent endpoints relate to placental development and function thymicylate denovo purine synthesis penovo 1 cnudecticles : (nucleotides metabolism 2 ~ folate 3 metabolic endpoints with different potential consequences donates methyl grps > - DNA methylation , his methyl ~ RNA methyl o small molecules Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Volume 737, Issues 1–2 2012 1 - 7 SAM CS- production of adenosyl methionine 3 C universal methyl donor 111 cells Folate is associated with placental- related pathologies Preeclampsia IUGR Placental abruption Spontaneous pregnancy loss 20  Preeclampsia (PE) Hypertensive disorder of pregnancy with placental origin Leading cause of fetal/maternal mortality/morbidity 1/20 pregnancies eclampsia (PE + seizures) Signs and symptoms New onset hypertension after 20 weeks GA Systolic > 140 mmHg and/or diastolic > 80 mmHg Proteinuria & excess protein urine) In Edema Lexcess fluidinbody's tissues Abnormal liver function HELLP Syndrome (Hemolysis, Elevated Liver enzymes, Low Platelet count) 21  Treatment No cure Delivery of placenta Etiology not completely understood – Placental hypoxia and damage – Excessive shedding of placental debris in maternal circulation – Maternal inflammation and endothelial dysfunction can Pare-eclampsia - > result in 7 only Offspring risks: PTB & FGR available treatment 2 since the is delivery Preterm birth – Severe PE requires delivery, may be earlier than 37 weeks GA Fetal growth restriction – Arteries carrying blood to placenta are affected – Fetus does not receive adequate blood/oxygen/nutrients – Slows growth 22  Long-term maternal risks Increased risk of vascular disease – Hypertension – Ischaemic heart disease – Stroke – Venous thromboembolism Increased risk of maternal mortality Bellamy (2007) BMJ 335(7627): 974. i poor placentation is part Preeclampsia of pathogenes is Normala o Invasive CTB (fetal origin) invade maternal spiral arteries S Leads to high-capacity dif. CTB has to vessels capable of endothelial epithelial providing placental > from - Fetal blood Vessel à > vascular Phenotype perfusion to sustain - mincry fetal growth CTB fail to adopt invasive endothelial phenotype Leads to poor/shallow ainvasion and low- por resistance maternal ↓ blood vessels X support increased Mohan (2013) J Evol Med Dent Sci 2:9283-9288 flow a fetal growth ↑ when resistance ↓ I pressure 23  (v supplement lower preventative inference > - & dds PE than no supp OR x observed in 15 or 3rd trimester ! (hypothesize question ) ? 2nd ↳ timing of trimester & CTB differentiation Cfolic acid supports - ↓ epi-endo Wen (2008) Am J Obstet Gynecol 198:45.e1-45.e7 ascreen ↓ Adverse pregnancy outcomes in women exposed to folic acid antagonists during pregnancy Methotrexate-treats RA /Eg : *Adjusted for year of birth, type of institution at birth, maternal age, parity, social assistance Wen (2008) CMAJ 179:1263-1268 24  Proposed mechanisms So does folic acid prevent PE? besity diabetes hypertension rish women : · , High , control treatment Cs 4 X dose FA ↳ standard treatment This trial is registered with NCT01355159 placebo Wen (2013) J Preg ID:294312 doi: 10.1155/2013/294312 25  /standard 5 mg the only country Canada - - > MTHFR - Polymorphism - 16 % ↳ ↓ MH snip avail ↳↑ ↳ Women w/MTHER & Bonip homosystein > - lower folate lower folate + higher homogys circulation as more responsive to supplem. D Wen (2018) BMJ 362:bmj.k3478 1 2 Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Volume 737, Issues 1–2 2012 1 - 7 Recall: FA affects placental growth and function 26  Placental invasion and growth Red-Horse (2004) JCI 114:744-754 Folate promotes trophoblast proliferation, viability and invasion 2 nm 20 nm 100 nm low dose normal dose high dose proliferation ↑ fa , a placental TB prolif , viability invasion knockout gene & & ⑧ viability invasion Moussa (2015) Br J Nutr 114:844-52 Ahmed (2016) Placenta 37:7-15 27  Folate deficiency induces apoptosis in human trophoblasts (folate in media) (folate free) (2005) BJOG 107:1513-1515 https://www.ptglab.com/news/blog/what-is-the-difference-between-necrosis-and-apoptosis/ folic acid -> all fun too Altered folate metabolism dysregulates TB progesterone secretion Moussa (2015) Br J Nutr 114:844-52 28  Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Volume 737, Issues 1–2 2012 1 - 7 Homocysteine is detrimental to placental health! Hcy is transported into syncytiotrophoblasts homosys-accum in stB ↑ Hcy could compete with other substrates of aa transporters, reducing aa for the placenta/fetus Induction of apoptosis in placenta? Affect placental metabolism? Activity of systems L and A in MVM are reduced in FGR Tsitsiou (2010) J Inherited Metab Dis 34:57-65 29  were complicated by IUGR. In both groups prior med- onsidered to be the 80th centile for ical or obstetric history was unremarkable. he third trimester of pregnancy. Homocysteine concentrations were signiWcantly 12 and folic acid blood was collected higher in the group of preeclamptic patients than the was centrifuged within 1 h. Plasma controls (P < 0.001) (Fig. 1). Mean levels of homocy- –8°C until the assay was performed steine in the preeclamptic group were 11.11 !mol/l and collection. in the control group were 6.40 !mol/l. measured with the Abbot Imx ion There were no diVerences between the groups r the quantitative measurement of regarding the levels of folic acid (P = 0.55) (Fig. 2). plasma on the Imx analyzer. Folate Mean values of folic acid in preeclamptic and control of 3.1–12.4 ng/ml are considered nor- women were 11.12 and 9.73 ng/ml, respectively. /ml are considered indeterminate and There were no diVerences between the groups re deWcient. Hcy is associated with increased risk for regarding the levels of vitamin B12 (P = 0.43) (Fig. 3). sured with a run on Imx system Park III). The Imx B12 assay is based pregnancy complications and adverse Mean values of vitamin B12 in the control group were 295.76 pg/ml, while in the preeclamptic group were icle Enzyme Immunoassay (MEIA). etween 223 and 1,132 pg/ml are nor- outcomes 356.15 pg/ml. Risk for vascular Hyperhocysteinemia - and 223 pg/ml are indeterminate and disease Foodsof PE w/higher 121 homocys compared eWcient. Uric acid was estimated in · Homocysteine level e enzymatic method (Trinder). most lowermost Choma 25 o to upper ysis with the t-test (SPSS Inc., Chi- cys] Concentrations in plasma (umol/L) was used for the comparison of the 20 homocysteine, folic acid and vitamin patients with preeclampsia and the 15 10 5 istics are shown in Table 1. Women a were more often primiparas and Control Preeclampsia éHcy also associated y cesarean section. In this group mean less and complications were more No change in levels of Fig. 1 Box-plot of homocysteine levels in preeclamptic patients and controls. Mean values are shown as a white line (P < 0.001) with higher OR of circulating folic acid or B12 prematurity, LBW, stillbirth, NTDs Makedos (2007) Arch Gynecol Obstet 275:121-124 Vollset (2000) Am J Clin Nutr 71:962-968 Hcy in maternal and umbilical cord associated with severe PE Concentrations in serum maternal serum fetal serum (2010) J Obstet Gyne Res 3:45-50 30  Hcy induces trophoblast apoptosis %TUNEL-positive nuclei FA mitigates the effect CON Hcy FA Hcy + FA (20 umol/l) (20 nmol/l) Di Simone (2004) Molec Hum Reprod 10:665-669 Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Volume 737, Issues 1–2 2012 1 - 7 3 31  Gestation-dependent changes in human placental global DNA methylation Demand for methyl gips for folate metabolism ↓ ↑ age gestation (2011) Molec Reprod Dev 78:150 Global DNA methylation changing Folic acid changes expression and methylation of imprinted genes in a dose-response manner - Inactive gene loss of imp. (no transcription) DNA methylation this of gene methylated CpG Coverexp) > - islands (H3K4) cancers + due DNA is inaccessible! to prometer switching X imprinted d paternally exp ~ on adults in a placenta tightly packed allele as all at prdit , di or histones L overexp. - > malignancies gene expression (heterochromatin) no FA mid FA dose high FA dose Rahat (2017) Scientific Reports 7:40774 32 Mom’s diet and/or genetics play a role in PE risk (and other pregnancy outcomes/pathologies) Placental implantation Placental cell Increased proliferation, homocysteine viability, invasion Maternal folate intake or metabolism Cell death Placental and/or viability and dysfunction function Mom’s diet and/or genetics play a role in PE risk (and other pregnancy outcomes/pathologies) SNPs – Single nucleotide polymorphism – Variation at a single point in a DNA sequence – May predict individual’s response to drugs, inherited disease susceptibility Vitamin B12/folate deficiency (+ related diseases) and dysfunctional transport can be predicted by > suscept to intera SNPs (B1] - FUT2 non-secretor status low > chronic autoimm disorder – FUT2 (SNP 772G>A) Population-specific phenotype driven by IS B12 two SNPs other infxn – TCN2 (SNP 776C>G) dystxnal - , Associated with susceptibility to certain resistance to transport infections and chronic autoimmune Cs influence qut microbial O fa – MTHFR (SNP 677C>T) low (BR2] diseases + resistance to certain infections comp +↑ IBS - Influences gut microbial composition & – RFC-1 (SNP 80A>G) ↑ risk cancra associated conditions (i.e. irritable bowel syndrome) Flow CFA) , cadascular disease Plays a role in infant gut microbial composition via breastmilk ↑ congenital dis When CFA] low to. begin us Wang (2001) Hum Mut 17:263-270 He (2017) Gen, Prot & Bioinfo 15(2): 94–100 Giampaoli (2020) Front Nutr 7:335 33  Folate & dads 2) highly verolit. cells ~ folate plays a part ? Freepik via Flaticon Folate deficiency and altered folate metabolism are associated with adverse outcomes for males Infertility and subfertility Reduced sperm number Increased sperm DNA damage Embryo defects Fertility: (2001) N Engl J Med 344:1172-3 (2005) Int J Androl 28:115-9 Sperm number: (2011) Fertil Steril 75:252-259 (2002) Fertil. Steril 77: 491–498 (2011) Pediatr Endocrinol Rev 8(Suppl 2):310-3 (2009) Mut Res 673:43-52 Sperm DNA damage: (2012) Mut Res 73:1-7 (2009) Fertil Steril 92:548-556 (2008) Hum Reprod 23:1014-22 (2009) Mut Res 673:43-52 (2008) Mut Res 655:59-67 Embryo defects: (2013) Nature Comm 4:2889 34  Folate deficiency results in reduced fertility in male mice Paternal folic acid deficiency ↳ ↓ embryo viability / # implantation but embryos lost during sites the same be birth of Preg fetus a term Fewer litters Similar implantation events More resorptions (embryo loss) MacFarlane (2017) unpublished Timing and mechanisms of embryo loss Guks ② spermatogenes is - Supplemented (6 mg folic acid/kg) Diet of male mice Males weaned onto & female -> on control deet 18mbs Control (2 mg folic acid/kg) diet-> > ↳ ensure sperm Deficient (0 mg folic acid/kg) fert eggs > - derived from Sperm progen Embryo analysis Breeding GD16.5 alls fully exposed Age (wks) 3wks 11 15 17 19 to deet F0 Sperm embryos C chose 3-" days be Weaned to diet Female mice fed control diet term > no - embryos > - male specimen loss bygod trimester collections MacFarlane (2017) unpub > - embryo observed > - Lose later or birth defect 35  no dif between diet ↳ sig of gips in terms of fertility > ↳ low pregrate (control -60% ) L Fertility and embryo loss did not differ amongst the diets at GD16.5 Diet (mg folic acid/kg diet) Deficient (0) Control (2) Supplemented (6) p n=8 n=9 n=8 Pregnancies (%/diet) 53.3 60 53.3 ns 1 Litter Size (#/female) 5.67 ± 0.73 5.00 ± 0.60 5.00 ± 0.54 ns2 Corpus Luteum (CL) (# CL/female) 8.11 ± 1.12 9.40 ± 0.56 9.13 ± 0.61 ns2 Implantation sites (#) 7.33 ± 0.82 7.60 ± 0.58 7.75 ± 0.82 ns2 Fertilisation rate (# implantation ns2 0.91 ± 0.05 0.81 ± 0.04 0.84 ± 0.05 sites/CL) Resorptions (# resorptions/female) 1.67 ± 0.24 2.60 ± 0.43 2.75 ± 0.84 ns2 Resorption rate (% embryos 30.0 ± 9.06 34.6 ± 5.36 32.8 ± 6.91 ns2 resorbed/female) 1 Chi square analysis 2 One-way ANOVA Tukey's HSD post-hoc analysis MacFarlane (2017) unpub GD16.5 embryo weight and length did not differ among the diets litters Embryo Weight Crown-Rump Diet (n) (mg) Length (mm) Deficient (0 mg/kg) 8 447 ± 29 14.4 ± 0.4 Control (2 mg/kg) 10 478 ± 34 15.0 ± 0.4 Supplemented (6 mg/kg 8 465 ± 5 14.9 ± 0.1 One-way ANOVA. Data are mean ± SEM MacFarlane (2017) unpub 36  Placental size dep. on dad's diet Supplementation resulted in larger placental weight and diameter at GD16.5 b b Placental Diameter (mm) 130 8.2 a,b Placental Weight (g) 125 8.1 120 a,b 8 7.9 a 115 a 7.8 110 7.7 105 7.6 100 7.5 95 7.4 0 2 6 0 2 6 Folic Acid (mg/kg) Folic Acid (mg/kg) Dose-response relationship One way ANOVA p - outside body gastroschisis ↳ small for embryos >- gestational age MacFarlane (2017) unpub C lack of intestinal wall closing potruding contents X enveloped in a sas 37  Paternal deficiency is associated with delayed development (SGA) and congenital defects GD16.5 fetuses SGA defects 25% of males ß 40% increase! produced a litter 50% of males Litters with SGA! C produced a litter smallgestation a with defects! >20% of embryos had defects! Fetuses * p≤0.04, Fisher’s exact test MacFarlane (2017) unpub Paternal deficiency delays skull ossification in GD 16.5 fetuses pr pr ipr fr ipr fr less colification ↓ Deficient Control Supplemented p-value1 n 50 48 39 Frontal (%) 90 100 100 TFR1 Crecep TFR1 -> clathrin coated pit Ferric ion -> Fe from dueo frame I dissociate ~ to fetal blood export from Sti throughF e export O Baropportin & basal mem of placental STB Fe -> FPN (placenta) dissec from Sangkhae (2019) Free Radic Biol Med 133:254-261. TFR1 > - bind to Fo TFRI complex transfered clathrin mediated via endocytosis 45 Iron in pregnancy: Early requirement to obtain in utero Breastmilk iron is poor source of iron for the Best infant Y – Delayed cord clamping to improve early iron transfer – Micronutrient supplementation pre-pregnancy Sprinkles Lucky iron fish Theculturetrip.com Could the gut microbiome play a role? Bacterial species in the gut require iron for redox reactions, metabolic pathways & survival ID and supplementation influence gut microbial comp