James Sebastian Martin Cuffe

Thyroid antibody positivity (TAb+) is common among reproductive‐aged women, and TAb+ in pregnancy is associated with complications including miscarriage and gestational diabetes. These associations persist in women with normal thyroid status, but there are minimal studies examining the underlying mechanisms. This study was designed to investigate the effects of TAb+ on maternal, placental and fetal health, with a focus on metabolism, using a Lewis rat model of TAb+ induced before pregnancy. Aspects of maternal fertility, cardiovascular adaptations and placental function were also assessed. Oestrous cycling was monitored prior to pregnancy, and an intraperitoneal glucose tolerance test was performed on embryonic day (E)16. Rats were killed at late pregnancy (E20), with maternal and fetal plasma and tissues collected for analysis of metabolic, endocrine and placental function. TAb+ rats did not have altered glucose tolerance during pregnancy but did have increased oestrous cycle length before pregnancy and altered concentrations of key reproductive hormones at E20, including rat placental lactogen. Placentas exposed to TAb+ were larger, with increased expression of glucose transporters and inflammatory markers. TAb+ dams had a reduced proportion of male fetuses per litter but no changes in litter size or fetal growth. This study demonstrates that although TAb+ was not associated with severe changes to the maternal parameters investigated, several aspects of placental biology were impacted. These findings support further investigation of TAb+ as a thyroid hormone‐independent contributor to pregnancy complications, with placental function emerging as a potential mechanistic target. image Key points: Thyroid antibody positivity (TAb+) is associated with an increased risk of complications during pregnancy, including miscarriage and gestational diabetes. Associations are often attributed to thyroid dysfunction, yet risks persist in euthyroid women. Research investigating the effects of TAb+ on glucose metabolism and placental function in models with normal thyroid hormone concentrations is required. In our rodent model of TAb+ during pregnancy, there were limited changes to glucose metabolism or litter size. However, we did see increased placental growth, altered rat placental lactogen and progesterone concentrations, and a reduced proportion of male fetuses. This study suggests the link between TAb+ and gestational diabetes is not driven by elevated antibodies. However, TAb+ impacts aspects of fertility, fetal survival and placental development, which has implications for other pregnancy disorders. The findings from this study highlight that thyroid antibodies are an important factor to consider when managing patients with TAb+ during pregnancy, outside of consequences to thyroid health.

Fetal growth restriction increases the risk of metabolic conditions such as gestational diabetes mellitus (GDM). One‐carbon metabolite and nutrient concentrations are dysregulated in GDM and influenced by antihyperglycaemic medications. However, it remains unclear whether disrupted one‐carbon metabolism contributes to GDM onset in growth restricted offspring and whether antihyperglycaemic medications exacerbate this dysregulation. We investigated the effects of growth restriction and antihyperglycaemic treatment on one‐carbon metabolism in pregnant rats and their fetuses. Uteroplacental insufficiency (Restricted) or sham (Control) surgery was performed on embryonic day (E)18 in Wistar–Kyoto rats. Female F1 offspring were mated and Restricted dams received daily metformin, insulin or vehicle from E13. Although restricted dams did not develop metabolic dysfunction during pregnancy, they exhibited reduced one‐carbon metabolism and a lower S‐adenosylmethionine:S‐adenosylhomocysteine (SAM:SAH) ratio, indicating reduced methylation capacity. These changes were exacerbated by both metformin and insulin. In F2 fetuses, plasma one‐carbon metabolites were unaffected despite changes in expression of genes involved in one‐carbon metabolism and DNA methylation in the placenta and fetal liver. F2 fetuses displayed an elevated pancreatic β‐cell:islet ratio. Antihyperglycaemic medications altered the expression of multiple one‐carbon metabolising enzymes in the maternal liver, the placenta junctional zone and the fetal liver. Metformin also increased pancreatic α‐cell area. This study suggests disrupted one‐carbon metabolism may underly programmed metabolic dysfunction and highlights the need for monitoring females born small. Both metformin and insulin induced similar physiological changes indicating that one is not safer than the other. Treatment decisions should consider potential impacts on long‐term health. image Key points: Being born growth restricted predisposes females to develop gestational diabetes (GDM) contributing to intergenerational transmission of disease. GDM is often treated with metformin or insulin. Disruptions to one‐carbon metabolism caused by growth restriction, insulin or metformin may explain transmission of disease. This study investigated the effects of growth restriction, metformin and insulin on one‐carbon metabolism and subsequent fetal outcomes in rats. Growth restriction impaired one‐carbon metabolism in mothers resulting in a reduced S‐adenosylmethionine:S‐adenosylhomocysteine ratio, changes to expression of associated enzymes in the placenta and fetal liver and an increase in the fetal β‐cell: pancreatic islet ratio. Metformin and insulin exacerbated deficits in one‐carbon metabolism in growth restricted dams and their fetuses, whereas metformin also increased pancreatic α‐cell area. This study demonstrates one‐carbon metabolism as a key regulator of programmed metabolic disease in pregnancy and informs about the appropriate treatment of GDM in women born growth restricted.

Background/Objectives: Dietary fibre promotes health, partly by mediating gut microbiota and short-chain fatty acid (SCFA) production. Pregnancy alters the relationship between dietary composition and the gut microbiota, and it is unclear if fibre intake during late pregnancy alters the abundance of SCFA bacteria and circulating SFCA concentrations. The aim of this study was to determine the impact of dietary fibre on faecal microbiome composition and circulating concentrations of SCFA acetate, butyrate, and propionate in late pregnancy. We also aimed to assess the impact of propionate treatment on placental function using cultured placental explants. Methods: 16S rRNA gene amplicon sequencing was performed on faecal DNA collected at 28 weeks of gestation from participants enrolled in the SPRING cohort study consuming a low or adequate fibre diet. Circualting SCFA were assessed. Placental explants were treated with sodium propionate. Results: Fibre intake did not impact microbial diversity or richness but did impact the abundance of specific bacterial genera. Pregnant participants with low-fibre diets had a greater abundance of Bacteroides and Sutterella, and dietary fibre intake (mg/day) negatively correlated with genera, including Sutterella, Bilophila, and Bacteroides. SCFA concentrations did not differ between groups but circulating concentrations of acetate, propionate, and butyrate did correlate with the abundance of key bacterial genera. Propionate treatment of placental explants did not alter mRNA expression of fatty acid receptors, antioxidants, or markers of apoptosis, nor did it impact pAMPK levels. Conclusions: This study demonstrates that the impact of dietary fibre on SCFA concentrations in pregnant women is modest, although this relationship may be difficult to discern given that other dietary factors differed between groups. Furthermore, this study demonstrates that propionate does not impact key pathways in placental tissue, suggesting that previous associations between this SCFA and placental dysfunction may be due to other maternal factors.

BACKGROUND: Prenatal alcohol exposure (PAE) around conception in preclinical models results in placental insufficiency, likely contributing to offspring abnormalities. Altered placental DNA methylation (DNAm) and gene expression suggest epigenetic mechanisms, perhaps involving impacts on methyl donor levels. PAE around conception in women is common but placental effects are rarely examined. This cohort study investigated associations between PAE around conception and intake/plasma measures of the methyl donors folate and choline, feto-placental blood flow, and placental growth measures, gene expression, and DNAm. METHODS: Pregnant participants delivered at Mater Mothers’ Hospital, Brisbane, Queensland, Australia ( n = 411). Dietary intake of choline and folate were calculated and plasma concentrations measured using mass spectrometry (MS) and clinical immunoanalyzer, respectively. Cerebroplacental ratio (CPR) was calculated using Doppler measurements. Placentas were weighed/measured at delivery and samples used to quantify methyl donors (MS), global DNAm (ELISA), and gene expression (quantitative PCR). Data were compared between control/abstinent and PAE groups, by fetal sex. RESULTS: A CPR S -adenosylmethionine and a trend toward increased DNAm. CONCLUSION: PAE around conception is associated with reduced CPR and altered placental growth measures, particularly in males, with potential implications for future health. FUNDING: National Health and Medical Research Council (APP1191217) and Mary McConnel Career Boost Program for Women in Paediatric Research (WIS132020).

Alcohol exposure during pregnancy disrupts fetal development and programs lifelong disease. We have shown, in rats, that alcohol exposure during the periconceptional period (PC:EtOH), causes placental dysfunction and cardiometabolic disease in offspring. The process of metabolising alcohol can cause oxidative stress and damage mitochondrial DNA (mtDNA). It is unknown whether alcohol metabolism in a rat model of PC:EtOH impacts oxidative stress markers and mitochondrial content in maternal and placental tissues. We aimed to determine whether PC:EtOH induced oxidative stress and reduced mtDNA in maternal liver and the placental labyrinth and junctional zone. Sprague–Dawley rats were given an ethanol liquid (12.5% v/v) or control (0%) diet for one oestrous cycle before mating to embryonic day (E) 4. Maternal livers were collected at E5 and E20. Placentas were collected at E20 and separated into the junctional zone and labyrinth zone. PC:EtOH reduced Cyp2e1 mRNA levels and mtDNA in the E5 liver with lower mtDNA persisting to E20, at which time mitochondrial proteins were also decreased. PC:EtOH also reduced mitochondrial content in the E20 junctional zone, although mitochondrial protein levels were unaffected. Superoxide dismutase activity was increased in the placental junctional zone and there was no evidence of oxidative stress. The present study demonstrates that alcohol exposure around conception, reduces mitochondrial content within the maternal liver and the junctional zone of the placenta towards the end of pregnancy. These prolonged deficits may have disrupted metabolic processes required for a healthy pregnancy. The study further supports avoiding alcohol when planning a pregnancy. image Key points: Even when alcohol is consumed only around conception (PC:EtOH), it can have profound impacts on the developing baby. Here, we use our established rat model to investigate if PC:EtOH causes oxidative stress and reduces mitochondrial content in the maternal liver immediately after exposure on embryonic day (E) 5. We also investigate these parameters at the end of pregnancy (E20) in maternal liver and the placenta. PC:EtOH reduced mitochondrial DNA content in the maternal liver by 77% at E5 and by 40% at E20. At E20, expression of proteins that form the electron transport chain were also reduced. The placenta had a more subtle reduction in mitochondrial DNA content, but protein levels of mitochondrial complexes were unchanged. There was no evidence of oxidative stress in the maternal liver or placenta in response to PC:EtOH. The lack of oxidative stress in the placenta may be a result of compensatory increases in antioxidants.

Linoleic acid (LA) is required for neuronal development. We have previously demonstrated sex-specific changes in cardiovascular and hepatic function in rat offspring from mothers consuming a high-LA diet, with some effects associated with reduced LA concentration in the postnatal diet. At this time, the impact of a high-maternal-LA diet on offspring brain development and the potential for the postnatal diet to alter any adverse changes are unknown. Rat offspring from mothers fed low- (LLA) or high-LA (HLA) diets during pregnancy and lactation were weaned at postnatal day 25 (PN25) and fed LLA or HLA diets until sacrifice in adulthood (PN180). In the offspring’s brains, the postnatal HLA diet increased docosapentaenoate in males. The maternal HLA diet increased LA, arachidonate, docosapentaenoate, C18:0 dimethylacetal (DMA), C16:0 DMA, C16:0 DMA/C16:0, and C18:0 DMA/C18:0, but decreased eoicosenoate, nervoniate, lignocerate, and oleate in males. Maternal and postnatal HLA diets reduced oleate and vaccenate and had an interaction effect on myristate, palmitoleate, and eicosapentaenoate in males. In females, maternal HLA diet increased eicosadienoate. Postnatal HLA diet increased stearate and docosapentaenoate. Maternal and postnatal HLA diets had an interaction effect on oleate, arachidate, and docosahexaenoic acid (DHA)/omega (n)-6 docosapentaenoic acid (DPA) in females. Postnatal HLA diet decreased DHA/n-6 DPA in males and females. Postnatal HLA diet increased plasma endocannabinoids (arachidonoyl ethanolamide and 2-arachidonoyl glycerol), as well as other N-acyl ethanolamides and testosterone. HLA diet alters brain fatty acids, plasma endocannabinoids, and plasmalogen concentrations in a development-specific and sex-specific manner.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is obtained from the maternal diet during pregnancy, and is essential for normal fetal growth and development. A maternal high-LA (HLA) diet alters maternal and offspring fatty acids, maternal leptin and male/female ratio at embryonic (E) day 20 (E20). We investigated the effects of an HLA diet on embryonic offspring renal branching morphogenesis, leptin signalling, megalin signalling and angiogenesis gene expression. Female Wistar Kyoto rats were fed low-LA (LLA; 1.44% energy from LA) or high-LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring were sacrificed and mRNA from kidneys was analysed by real-time PCR. Maternal HLA decreased the targets involved in branching morphogenesis Ret and Gdnf in offspring, independent of sex. Furthermore, downstream targets of megalin, namely mTOR, Akt3 and Prkab2, were reduced in offspring from mothers consuming an HLA diet, independent of sex. There was a trend of an increase in the branching morphogenesis target Gfra1 in females (p = 0.0517). These findings suggest that an HLA diet during pregnancy may lead to altered renal function in offspring. Future research should investigate the effects an HLA diet has on offspring kidney function in adolescence and adulthood.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is essential for fetal growth and development. A maternal high LA (HLA) diet alters cardiovascular development in adolescent rats and hepatic function in adult rats in a sex-specific manner. We investigated the effects of an HLA diet on adolescent offspring hepatic lipids and hepatic lipid metabolism gene expression, and the ability of the postnatal diet to alter these effects. Female Wistar Kyoto rats were fed low LA (LLA; 1.44% energy from LA) or high LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring, weaned at postnatal day (PN) 25, were fed LLA or HLA and euthanised at PN40 (n = 6–8). Maternal HLA increased circulating uric acid, decreased hepatic cholesterol and increased hepatic Pparg in males, whereas only hepatic Srebf1 and Hmgcr increased in females. Postnatal (post-weaning) HLA decreased liver weight (% body weight) and increased hepatic Hmgcr in males, and decreased hepatic triglycerides in females. Maternal and postnatal HLA had an interaction effect on Lpl, Cpt1a and Pparg in females. These findings suggest that an HLA diet both during and after pregnancy should be avoided to improve offspring disease risk.

Alterations in the hypothalamic–pituitary–adrenal (HPA) axis and associated changes in circulating levels of glucocorticoids are integral to an organism’s response to stressful stimuli. Glucocorticoids acting via glucocorticoid receptors (GRs) play a role in fertility, reproduction, placental function, and foetal development. GRs are ubiquitously expressed throughout the female reproductive system and regulate normal reproductive function. Stress-induced glucocorticoids have been shown to inhibit reproduction and affect female gonadal function by suppressing the hypothalamic–pituitary–gonadal (HPG) axis at each level. Furthermore, during pregnancy, a mother’s exposure to prenatal stress or external glucocorticoids can result in long-lasting alterations to the foetal HPA and neuroendocrine function. Several GR isoforms generated via alternative splicing or translation initiation from the GR gene have been identified in the mammalian ovary and uterus. The GR isoforms identified include the splice variants, GRα and GRβ, and GRγ and GR-P. Glucocorticoids can exert both stimulatory and inhibitory effects and both pro- and anti-inflammatory functions in the ovary, in vitro. In the placenta, thirteen GR isoforms have been identified in humans, guinea pigs, sheep, rats, and mice, indicating they are conserved across species and may be important in mediating a differential response to stress. Distinctive responses to glucocorticoids, differential birth outcomes in pregnancy complications, and sex-based variations in the response to stress could all potentially be dependent on a particular GR expression pattern. This comprehensive review provides an overview of the structure and function of the GR in relation to female fertility and reproduction and discusses the changes in the GR and glucocorticoid signalling during pregnancy. To generate this overview, an extensive non-systematic literature search was conducted across multiple databases, including PubMed, Web of Science, and Google Scholar, with a focus on original research articles, meta-analyses, and previous review papers addressing the subject. This review integrates the current understanding of GR variants and their roles in glucocorticoid signalling, reproduction, placental function, and foetal growth.