Deciphering the role of the gut microbiota in murine MASH and MASH-to-HCC transition

Having reached an epidemic magnitude, obesity has experienced a drastic increase in prevalence over the past decades. Together with obesity, associated pathologies, including metabolic syndrome, became more common, which further increase the risk of long-term complications. As such, chronic manifestation of these conditions frequently leads to metabolic dysfunction-associated steatohepatitis (MASH). MASH has gained significantly in importance due to the lack of treatment options and the progression towards fibrosis and hepatocellular carcinoma (HCC), making it the fastest-growing cause of primary liver cancer. The intestinal microbiota is known to be influenced by a variety of metabolic diseases and to actively contribute to inflammatory events. Indications for a role of the microbiota in chronic liver diseases exist, but if and how intestinal bacteria drive the progression of MASH towards HCC remains uncertain. To investigate the role of the microbiota in choline-deficient high fat diet (CD-HFD)-induced MASH and HCC I depleted intestinal bacteria using a broad-spectrum antibiotics (ABx) cocktail. To decipher the effect of microbiota depletion on different stages of pathogenesis, I performed ABx treatment either prophylactically or therapeutically after disease onset. I analyzed the effect of microbiota depletion on MASH and HCC pathogenesis by characterizing immunological, transcriptional and metabolic signatures in the mice. To evaluate the effects of CD-HFD feeding and the ABx treatment on intestinal bacteria, I determined the microbiota composition by shotgun metagenomic sequencing. My data confirmed that the CD-HFD MASH model can recapitulate previously described mechanisms involving the gut-liver axis and that ABx-mediated microbiota depletion reduces MASH development. Moreover, I was able to show that ABx treatment limits MASH-to-HCC transition in CD-HFD mice by preventing fibrosis development, which resulted in a lower liver tumor incidence. In addition, prophylactic ABx treatment modified certain tumor characteristics, which were possibly linked to an increased tumor size observed in these mice. Therapeutic ABx treatment reversed this effect, indicating an ambivalent role of microbial presence at different stages of the pathogenesis. Furthermore, I found diminished IgA-dependent activation of FCGR1-expressing myeloid cells as the likely cause of ABx-mediated reduced fibrogenesis, which was presumably supported by a variety of metabolic and transcriptional changes upon ABx treatment with additional beneficial effects on MASH-to-HCC transition. Although further research is needed to understand the spectrum of underlying mechanisms, my study confirms the detrimental contributions of intestinal bacteria to the MASH-to-HCC transition and suggests an ambivalent role of bacteria depending on the disease stage

Epitranscriptomic regulation in breast cancer and PCB-induced liver disease.

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, whereas knockdown of A2B1 in ET-resistant cells restored ET-sensitivity. microRNAs (miRNAs) downregulated by transient overexpression of A2B1 were identified to target two key enzymes (PSAT1 and PHGDH) in the serine biosynthetic pathway (SSP) which is upregulated in ET-resistant BCa cells and in tumors from patients with ET-resistant disease. Using luciferase assays, PSAT1 and PHGDH were validated as bona fide targets of miRNAs downregulated by A2B1 (miR-145-5p and miR-424-5p targeting PSAT1, miR-34b-5p and miR-876-5p targeting PHGDH). Exogenous overexpression of the validated miRNAs decreased endogenous PSAT1 and PHGDH in ET-resistant BCa cells, resulting in increased sensitivity to ET in vitro. In the second model, alterations in the m6A epitranscriptome were identified in the livers of male C57Bl/6Jmice after a single, oral exposure to polychlorinated biphenyls (PCB), a class of persistent organic pollutants, in combination with 12 weeks on a high fat diet (HFD). Our results demonstrated that exposure to PCBs in combination with a HFD resulted in major changes to the mRNA and miRNA transcriptomes, and m6A epitranscriptome. Pathway analysis of the genes in which m6A peaks were altered identified pathways involved in the progression from steatosis to steatohepatitis in NAFLD. PCB exposures also resulted in changes to alternative splicing (AS) mechanisms and events, suggesting that PCB-induced m6A changes contribute to altered isoforms expression in NAFLD. Taken together, the results in this dissertation demonstrate the significant role of altered m6A in two common human diseases

One-carbon metabolism and epigenetic programming of mammalian development

One-carbon (1C) metabolism comprises a series of integrated metabolic pathways, including the linked methionine-folate cycles, that provide methyl groups for the synthesis of biomolecules and the epigenetic regulation of gene expression via chromatin methylation. Most of the research investigating the function of 1C metabolism pertains to studies undertaken in the rodent liver. Comparatively little is known about the function of 1C metabolism in reproductive and embryonic cells, particularly in domestic ruminant species. Periconceptional dietary deficiencies in 1C substrates and cofactors are known to lead to epigenetic alterations in DNA methylation in genes that regulate key developmental processes in the embryo. Such modifications can have negative implications on the subsequent development, metabolism and health of offspring. This thesis sought to improve current understanding of the regulation of 1C metabolism in the ruminant liver, ovary and preimplantation embryo through in vivo and in vitro nutritional supplementation experiments coupled with metabolomic, transcriptomic and epigenetic analyses. The first part of this thesis (Chapter 2) assessed the metabolic consequences of dietary methyl deficiency using novel mass spectrometry–based methods that were developed for the quantification of B vitamins, folates and 1C-related amines in sheep liver. This study provided the first comparison of the relative abundance of bioactive 1C metabolites in liver harvested from methyl deficient sheep relative to a control study population of abattoir derived sheep. Relevant reductions in dietary methyl availability led to significant alterations in hepatic 1C metabolite concentrations. Large natural variations in the hepatic concentrations of individual metabolites in both sheep study populations reflected the dietary and genetic variation in our chosen outbred model species. These metabolomics platforms will be useful for investigating 1C metabolism and linked biochemical pathways in order to facilitate future dietary and genetic studies of metabolic health and epigenetic regulation of gene expression. Based on the absence of methionine cycle enzyme transcripts (e.g. MAT1A and BHMT) in the bovine ovary and preimplantation embryo, the second part of this thesis (Chapter 3 and Chapter 4) addressed the hypothesis that ruminant reproductive and embryonic cells are highly sensitive to methyl group availability and, therefore, epigenetic programming during the periconceptional period. Transcript analyses confirmed MAT2A expression in the bovine liver, ovary and at each stage of preimplantation embryo development assessed to Day 8. Transcripts for BHMT isoforms (BHMT and BHMT2) were detected in the bovine ovary but were weak or absent in embryos, highlighting a key difference in methionine metabolism between hepatic and reproductive cells. Bovine embryos were produced in vitro using custom-made media containing 0 (nonphysiological), 10 (low physiological), 50 (high physiological), and 500 µmol/L (supraphysiological) added methionine (Chapter 3). Gross morphological assessments of embryo stage, grade, cell lineage allocation and primary sex ratio revealed that culture in non- and supraphysiological methionine concentrations was detrimental for embryo development, whilst culture in the high physiological concentration appeared to be best. Reduced representation bisulphite sequencing (RRBS) of inner cell mass (ICM) and trophectoderm (TE) cells immunodissected from Day 8 blastocysts demonstrated that culturing embryos in low physiological methionine led to global hypomethylation within both cell lineages. Bioinformatic analyses of differentially methylated genes included gene set enrichment analyses (GSEA). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched within the ICM were associated with protein catabolism and autophagy, and significant terms and pathways enriched within the TE were associated with cellular transport. Of particular biological interest was the loss of methylation within regulatory region (DMR2) of the paternally imprinted gene, IGF2R, in the TE following culture in low physiological methionine. Transcript analysis found no significant effect of methionine concentration on the expression of IGF2R or the antisense transcript, AIRN, in the primary cell lineages of the Day 8 bovine preimplantation embryo. Hypomethylation of IGF2R DMR2 has been associated with aberrant IGF2R expression and large offspring syndrome (LOS) in cattle and sheep that were subjected to embryo manipulation during assisted reproductive technology (ART) procedures, such as somatic cell nuclear transfer (SCNT) or non-physiological in vitro embryo culture environments. Chapter 5 sought to evaluate the effect of somatic donor cell type on epigenetic reprogramming via DNA methylation in hepatocytes isolated from cloned sheep. RRBS facilitated the comparison of methylation reprogramming between Finn Dorset (D) clone hepatocytes and their mammary epithelial (OP5) donor cell line; and, Lleyn (L) clone hepatocytes and their Lleyn fetal fibroblast (LFF4) donor cell line. Methylation was most closely correlated between D and L clone hepatocytes than between clones and their respective donor cell lines. In general, hepatocytes were hypomethylated relative to their somatic donor cell nuclei. GSEA identified genes that encoded transcription factor proteins enriched within the ‘Sequence-specific DNA binding’ term (GO:0043565) as differentially methylated between clone hepatocytes and their donor cell lines. In addition, imprinted genes, including IGF2R, were differentially methylated in clone hepatocytes relative to somatic cell nuclei. In summary, this thesis promotes and supports the importance of an optimal methyl balance to support periconceptional development in mammals. The experiments detailed herein provide an insight into the metabolic consequences of dietary methyl deficiency (and excess) in outbred populations of domestic ruminants, with a specific focus on the liver, ovary and preimplantation embryo. The results demonstrate that tissue- and species-specific features of 1C metabolism render ruminant embryonic cells sensitive to methionine inputs within a physiological range. The observation that in vitro embryo culture and manipulation techniques, such as somatic cell nuclear transfer, can cause epigenetic alterations to DNA methylation during preimplantation development provides a basis for further study into the safety and efficacy of emerging assisted reproductive technologies

One-carbon metabolism and epigenetic programming of mammalian development

One-carbon (1C) metabolism comprises a series of integrated metabolic pathways, including the linked methionine-folate cycles, that provide methyl groups for the synthesis of biomolecules and the epigenetic regulation of gene expression via chromatin methylation. Most of the research investigating the function of 1C metabolism pertains to studies undertaken in the rodent liver. Comparatively little is known about the function of 1C metabolism in reproductive and embryonic cells, particularly in domestic ruminant species. Periconceptional dietary deficiencies in 1C substrates and cofactors are known to lead to epigenetic alterations in DNA methylation in genes that regulate key developmental processes in the embryo. Such modifications can have negative implications on the subsequent development, metabolism and health of offspring. This thesis sought to improve current understanding of the regulation of 1C metabolism in the ruminant liver, ovary and preimplantation embryo through in vivo and in vitro nutritional supplementation experiments coupled with metabolomic, transcriptomic and epigenetic analyses. The first part of this thesis (Chapter 2) assessed the metabolic consequences of dietary methyl deficiency using novel mass spectrometry–based methods that were developed for the quantification of B vitamins, folates and 1C-related amines in sheep liver. This study provided the first comparison of the relative abundance of bioactive 1C metabolites in liver harvested from methyl deficient sheep relative to a control study population of abattoir derived sheep. Relevant reductions in dietary methyl availability led to significant alterations in hepatic 1C metabolite concentrations. Large natural variations in the hepatic concentrations of individual metabolites in both sheep study populations reflected the dietary and genetic variation in our chosen outbred model species. These metabolomics platforms will be useful for investigating 1C metabolism and linked biochemical pathways in order to facilitate future dietary and genetic studies of metabolic health and epigenetic regulation of gene expression. Based on the absence of methionine cycle enzyme transcripts (e.g. MAT1A and BHMT) in the bovine ovary and preimplantation embryo, the second part of this thesis (Chapter 3 and Chapter 4) addressed the hypothesis that ruminant reproductive and embryonic cells are highly sensitive to methyl group availability and, therefore, epigenetic programming during the periconceptional period. Transcript analyses confirmed MAT2A expression in the bovine liver, ovary and at each stage of preimplantation embryo development assessed to Day 8. Transcripts for BHMT isoforms (BHMT and BHMT2) were detected in the bovine ovary but were weak or absent in embryos, highlighting a key difference in methionine metabolism between hepatic and reproductive cells. Bovine embryos were produced in vitro using custom-made media containing 0 (nonphysiological), 10 (low physiological), 50 (high physiological), and 500 µmol/L (supraphysiological) added methionine (Chapter 3). Gross morphological assessments of embryo stage, grade, cell lineage allocation and primary sex ratio revealed that culture in non- and supraphysiological methionine concentrations was detrimental for embryo development, whilst culture in the high physiological concentration appeared to be best. Reduced representation bisulphite sequencing (RRBS) of inner cell mass (ICM) and trophectoderm (TE) cells immunodissected from Day 8 blastocysts demonstrated that culturing embryos in low physiological methionine led to global hypomethylation within both cell lineages. Bioinformatic analyses of differentially methylated genes included gene set enrichment analyses (GSEA). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched within the ICM were associated with protein catabolism and autophagy, and significant terms and pathways enriched within the TE were associated with cellular transport. Of particular biological interest was the loss of methylation within regulatory region (DMR2) of the paternally imprinted gene, IGF2R, in the TE following culture in low physiological methionine. Transcript analysis found no significant effect of methionine concentration on the expression of IGF2R or the antisense transcript, AIRN, in the primary cell lineages of the Day 8 bovine preimplantation embryo. Hypomethylation of IGF2R DMR2 has been associated with aberrant IGF2R expression and large offspring syndrome (LOS) in cattle and sheep that were subjected to embryo manipulation during assisted reproductive technology (ART) procedures, such as somatic cell nuclear transfer (SCNT) or non-physiological in vitro embryo culture environments. Chapter 5 sought to evaluate the effect of somatic donor cell type on epigenetic reprogramming via DNA methylation in hepatocytes isolated from cloned sheep. RRBS facilitated the comparison of methylation reprogramming between Finn Dorset (D) clone hepatocytes and their mammary epithelial (OP5) donor cell line; and, Lleyn (L) clone hepatocytes and their Lleyn fetal fibroblast (LFF4) donor cell line. Methylation was most closely correlated between D and L clone hepatocytes than between clones and their respective donor cell lines. In general, hepatocytes were hypomethylated relative to their somatic donor cell nuclei. GSEA identified genes that encoded transcription factor proteins enriched within the ‘Sequence-specific DNA binding’ term (GO:0043565) as differentially methylated between clone hepatocytes and their donor cell lines. In addition, imprinted genes, including IGF2R, were differentially methylated in clone hepatocytes relative to somatic cell nuclei. In summary, this thesis promotes and supports the importance of an optimal methyl balance to support periconceptional development in mammals. The experiments detailed herein provide an insight into the metabolic consequences of dietary methyl deficiency (and excess) in outbred populations of domestic ruminants, with a specific focus on the liver, ovary and preimplantation embryo. The results demonstrate that tissue- and species-specific features of 1C metabolism render ruminant embryonic cells sensitive to methionine inputs within a physiological range. The observation that in vitro embryo culture and manipulation techniques, such as somatic cell nuclear transfer, can cause epigenetic alterations to DNA methylation during preimplantation development provides a basis for further study into the safety and efficacy of emerging assisted reproductive technologies

Role of adipose tissue in the pathogenesis and treatment of metabolic syndrome

© Springer International Publishing Switzerland 2014. Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is not only the primary disease of fat cells, but also a major risk factor for the development of Type 2 diabetes, cardiovascular disease, hypertension, and metabolic syndrome (MetS). Today, adipocytes and adipose tissue are no longer considered passive participants in metabolic pathways. In addition to storing lipid, adipocytes are highly insulin sensitive cells that have important endocrine functions. Altering any one of these functions of fat cells can result in a metabolic disease state and dysregulation of adipose tissue can profoundly contribute to MetS. For example, adiponectin is a fat specific hormone that has cardio-protective and anti-diabetic properties. Inhibition of adiponectin expression and secretion are associated with several risk factors for MetS. For this purpose, and several other reasons documented in this chapter, we propose that adipose tissue should be considered as a viable target for a variety of treatment approaches to combat MetS

Defining the impact of paternal diet on maternal cardiometabolic ill-health in late gestation

It is well-established that poor maternal nutrition prior to, or during, pregnancy can have a range of adverse consequences for the health of the mother and her offspring. For the mother, these consequences can become manifest in an increased risk of developing conditions such as preeclampsia and gestational diabetes. For her offspring, studies have shown an increased predisposition towards a series of non-communicable diseases including obesity, type-2 diabetes, and cardiovascular disease. Such connections between maternal health and offspring well-being have been defined over recent decades through the detailed examination of human epidemiological cohorts as well as a multitude of animal models. While the association between poor maternal health and perturbed offspring development is widely acknowledged, the role that a father plays in directing the development and long-term health of his offspring has been overlooked. However, it is becoming increasingly evident that the father plays a more significant role than just contributing to his progeny’s genome. Similar to the established maternal programming studies, the use of historical epidemiological data sets, supported by mechanistic animal models, reveals a complex process through which poor paternal health influences post-fertilization development and offspring well-being. Such observations have underpinned a new field within the Developmental Origins of Health and Disease (DOHaD) hypothesis, known as the Paternal Origins of Health and Disease (POHaD). In particular, studies have focused on the role of paternal nutrition, being either increased or decreased in specific macro- and micro-nutrients including protein, fat, sugar and folate. Through these studies, a complex system involving perturbed testicular function and spermatogenesis, epididymal maturation and exosomal modifications, sperm epigenetic status and DNA integrity as well as seminal plasma composition have all been identified as mediators of paternal programming. Therefore, this thesis aimed to investigate the impact of a paternal suboptimal diet on male physiology, fetal development, and late gestation maternal cardio-metabolic health. Furthermore, as altered sperm epigenetic status has been identified as one central programming mechanism, the impact of methyl-donor supplementation was also investigated to determine if this could negate the detrimental effects of the suboptimal diets. Additionally, as poor maternal health in pregnancy is a significant risk factor for altered fetal development, I also assessed the impact of paternal diet on maternal cardio-metabolic status in late gestation. To achieve these aims, male C57/BL6J mice were fed one of the five diets; a control diet (CD, 18% casein, 21% sugar, 10% fat), low protein diet (LPD, 9% casein, 24% sugar, 10% fat), Western diet (WD, 19% casein, 34% sugar, 21% fat), LPD supplemented with methyl donors (MDLPD, 15% D, L-methionine, 7.5% betaine, 5% choline chloride) or WD supplemented with methyl donors (MDWD) for a minimum of 7 weeks. Males were mated with chow-fed, virgin 8–12-week-old C57BL/6J females. At embryonic day 17.5, dams were culled for the collection of a range of maternal and fetal tissues. At the end of the study, stud males were then culled for the analysis of male organ size. I observed that male weight was not altered in response to being fed any of the experimental diets. However, I did observe differential sizing of several organs, especially within the WD and MDWD males. Of note, was an increase in gonadal fat in these males, indicating that while total body weight was not altered, adiposity appeared to increase. Following mating, there were no detrimental impacts of any diet on male fertility, as determined by late gestation litter size. While fetal growth and organ sizing appeared largely unaffected by the paternal diets, analysis of the fetal cardiac transcriptome revealed altered expression of multiple genes. In LPD and MDL fetuses, I observed differential expression of genes within central lipid, amino acid, and carbohydrate metabolic processes as well as cardiovascular disease pathways. In contrast, WD hearts demonstrated upregulation of genes involved in angiogenesis, embryonic organ development, and lipid metabolism as well as an abnormal heart morphology phenotype. Analysis of placental morphology and gene expression revealed no difference in overall morphology or sizing of the labyrinth (Lz) or junctional (Jz) zones. Using Masson’s Trichrome staining to visualize the amount of connective tissue within the placenta, revealed a significant reduction in the amount of overall staining within WD and MDWD placentas. Furthermore, I observed reduced expression of genes central within the renin-angiotensin system (RAS), apoptosis and one-carbon metabolism pathways within WD and MDWD placentas. Finally, analysis of maternal cardiometabolic health in late gestation showed there to be minimal changes in serum and hepatic metabolites, gut microbiota or mediators of cardiovascular function in response to paternal diet. These observations are of interest to human health as they demonstrate that in pregnancies uncomplicated by male infertility, or perturbed by maternal metabolic health, fetal cardiovascular programming still occurred. Furthermore, changes in fetal cardiac gene expression occurred in response to both a relatively moderate (LPD) and severe (WD) paternal dietary challenge. Further studies are required to define the underlying mechanisms through which poor paternal diet impacts fetal heart gene expression and the consequences of these changes for long-term offspring health

Defining the impact of paternal diet on maternal cardiometabolic ill-health in late gestation

It is well-established that poor maternal nutrition prior to, or during, pregnancy can have a range of adverse consequences for the health of the mother and her offspring. For the mother, these consequences can become manifest in an increased risk of developing conditions such as preeclampsia and gestational diabetes. For her offspring, studies have shown an increased predisposition towards a series of non-communicable diseases including obesity, type-2 diabetes, and cardiovascular disease. Such connections between maternal health and offspring well-being have been defined over recent decades through the detailed examination of human epidemiological cohorts as well as a multitude of animal models. While the association between poor maternal health and perturbed offspring development is widely acknowledged, the role that a father plays in directing the development and long-term health of his offspring has been overlooked. However, it is becoming increasingly evident that the father plays a more significant role than just contributing to his progeny’s genome. Similar to the established maternal programming studies, the use of historical epidemiological data sets, supported by mechanistic animal models, reveals a complex process through which poor paternal health influences post-fertilization development and offspring well-being. Such observations have underpinned a new field within the Developmental Origins of Health and Disease (DOHaD) hypothesis, known as the Paternal Origins of Health and Disease (POHaD). In particular, studies have focused on the role of paternal nutrition, being either increased or decreased in specific macro- and micro-nutrients including protein, fat, sugar and folate. Through these studies, a complex system involving perturbed testicular function and spermatogenesis, epididymal maturation and exosomal modifications, sperm epigenetic status and DNA integrity as well as seminal plasma composition have all been identified as mediators of paternal programming. Therefore, this thesis aimed to investigate the impact of a paternal suboptimal diet on male physiology, fetal development, and late gestation maternal cardio-metabolic health. Furthermore, as altered sperm epigenetic status has been identified as one central programming mechanism, the impact of methyl-donor supplementation was also investigated to determine if this could negate the detrimental effects of the suboptimal diets. Additionally, as poor maternal health in pregnancy is a significant risk factor for altered fetal development, I also assessed the impact of paternal diet on maternal cardio-metabolic status in late gestation. To achieve these aims, male C57/BL6J mice were fed one of the five diets; a control diet (CD, 18% casein, 21% sugar, 10% fat), low protein diet (LPD, 9% casein, 24% sugar, 10% fat), Western diet (WD, 19% casein, 34% sugar, 21% fat), LPD supplemented with methyl donors (MDLPD, 15% D, L-methionine, 7.5% betaine, 5% choline chloride) or WD supplemented with methyl donors (MDWD) for a minimum of 7 weeks. Males were mated with chow-fed, virgin 8–12-week-old C57BL/6J females. At embryonic day 17.5, dams were culled for the collection of a range of maternal and fetal tissues. At the end of the study, stud males were then culled for the analysis of male organ size. I observed that male weight was not altered in response to being fed any of the experimental diets. However, I did observe differential sizing of several organs, especially within the WD and MDWD males. Of note, was an increase in gonadal fat in these males, indicating that while total body weight was not altered, adiposity appeared to increase. Following mating, there were no detrimental impacts of any diet on male fertility, as determined by late gestation litter size. While fetal growth and organ sizing appeared largely unaffected by the paternal diets, analysis of the fetal cardiac transcriptome revealed altered expression of multiple genes. In LPD and MDL fetuses, I observed differential expression of genes within central lipid, amino acid, and carbohydrate metabolic processes as well as cardiovascular disease pathways. In contrast, WD hearts demonstrated upregulation of genes involved in angiogenesis, embryonic organ development, and lipid metabolism as well as an abnormal heart morphology phenotype. Analysis of placental morphology and gene expression revealed no difference in overall morphology or sizing of the labyrinth (Lz) or junctional (Jz) zones. Using Masson’s Trichrome staining to visualize the amount of connective tissue within the placenta, revealed a significant reduction in the amount of overall staining within WD and MDWD placentas. Furthermore, I observed reduced expression of genes central within the renin-angiotensin system (RAS), apoptosis and one-carbon metabolism pathways within WD and MDWD placentas. Finally, analysis of maternal cardiometabolic health in late gestation showed there to be minimal changes in serum and hepatic metabolites, gut microbiota or mediators of cardiovascular function in response to paternal diet. These observations are of interest to human health as they demonstrate that in pregnancies uncomplicated by male infertility, or perturbed by maternal metabolic health, fetal cardiovascular programming still occurred. Furthermore, changes in fetal cardiac gene expression occurred in response to both a relatively moderate (LPD) and severe (WD) paternal dietary challenge. Further studies are required to define the underlying mechanisms through which poor paternal diet impacts fetal heart gene expression and the consequences of these changes for long-term offspring health