Genetic insights into biological mechanisms governing human ovarian ageing.

Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Maternal antioxidant treatment protects adult offspring against memory loss and hippocampal atrophy in a rodent model of developmental hypoxia.

Chronic fetal hypoxia is one of the most common outcomes in complicated pregnancy in humans. Despite this, its effects on the long-term health of the brain in offspring are largely unknown. Here, we investigated in rats whether hypoxic pregnancy affects brain structure and function in the adult offspring and explored underlying mechanisms with maternal antioxidant intervention. Pregnant rats were randomly chosen for normoxic or hypoxic (13% oxygen) pregnancy with or without maternal supplementation with vitamin C in their drinking water. In one cohort, the placenta and fetal tissues were collected at the end of gestation. In another, dams were allowed to deliver naturally, and offspring were reared under normoxic conditions until 4 months of age (young adult). Between 3.5 and 4 months, the behavior, cognition and brains of the adult offspring were studied. We demonstrated that prenatal hypoxia reduced neuronal number, as well as vascular and synaptic density, in the hippocampus, significantly impairing memory function in the adult offspring. These adverse effects of prenatal hypoxia were independent of the hypoxic pregnancy inducing fetal growth restriction or elevations in maternal or fetal plasma glucocorticoid levels. Maternal vitamin C supplementation during hypoxic pregnancy protected against oxidative stress in the placenta and prevented the adverse effects of prenatal hypoxia on hippocampal atrophy and memory loss in the adult offspring. Therefore, these data provide a link between prenatal hypoxia, placental oxidative stress, and offspring brain health in later life, providing insight into mechanism and identifying a therapeutic strategy.

Exploring Telomere Dynamics in Aging Male Rat Tissues: Can Tissue-Specific Differences Contribute to Age-Associated Pathologies?

INTRODUCTION: Due to increasing lifespan, global aging rates are rising rapidly and age-associated diseases are increasing. To ensure that health span is concomitant with life span, a greater understanding of cellular mechanisms of aging is important. METHODS: Telomere length analysis from a wide range of tissues from weaning, young adult, and middle-aged (3, 12 and 52 week) male Wistar rats were conducted using Southern blotting. Telomere lengths were compared between tissues and ages using regression models based on the ratios of longest-to-shortest telomere fragments. RESULTS: Robust linear age-dependent telomere attrition was observed in the liver; 3 versus 12 weeks, 3 versus 52 weeks (p < 0.01), 12 versus 52 weeks (p < 0.05) and the heart; 3 versus 12 weeks (p < 0.05) and 3 versus 52 weeks (p < 0.001). More subtle shortening was observed in aorta and epididymal fat; 3 and 12 versus 52 weeks (p < 0.001) and in skeletal muscle; 3 versus 52 weeks (p < 0.05), 12 versus 52 weeks (p < 0.01). Young thymus telomeres increased in length (3 vs. 12 weeks) and then shortened between 12 and 52 weeks (p < 0.001). We also reported disparity in telomere shortening within tissues: telomeres in aging brain cortex significantly shortened; 3 versus 52 weeks (p < 0.05), 12 versus 52 weeks (p < 0.01). This was not seen in the hypothalamic region. A robust stepwise shortening was observed in the renal cortex; 3 versus 12 weeks, 12 versus 52 weeks (p < 0.05), and 3 versus 52 weeks (p < 0.001), which was not as apparent in the renal medulla; 3 versus 12 weeks (p < 0.01) and 3 versus 52 weeks (p < 0.01). The vastus lateralis skeletal muscle demonstrated the shortest telomere length at weaning and underwent robust age-associated attrition; 3 versus 52 weeks (p < 0.05), 12 versus 52 weeks (p < 0.01). We demonstrated that specific tissues exhibit unique telomere attrition profiles which may partially explain why certain diseases are more prevalent in aged individuals. DISCUSSION/CONCLUSION: We show wide variations between tissues in vulnerability to the aging process. In the future, this may help target potential interventions to improve health span.

Comparative impact of pharmacological treatments for gestational diabetes on neonatal anthropometry independent of maternal glycaemic control: A systematic review and meta-analysis.

BACKGROUND: Fetal growth in gestational diabetes mellitus (GDM) is directly linked to maternal glycaemic control; however, this relationship may be altered by oral anti-hyperglycaemic agents. Unlike insulin, such drugs cross the placenta and may thus have independent effects on fetal or placental tissues. We investigated the association between GDM treatment and fetal, neonatal, and childhood growth. METHODS AND FINDINGS: PubMed, Ovid Embase, Medline, Web of Science, ClinicalTrials.gov, and Cochrane databases were systematically searched (inception to 12 February 2020). Outcomes of GDM-affected pregnancies randomised to treatment with metformin, glyburide, or insulin were included. Studies including preexisting diabetes or nondiabetic women were excluded. Two reviewers independently assessed eligibility and risk of bias, with conflicts resolved by a third reviewer. Maternal outcome measures were glycaemic control, weight gain, and treatment failure. Offspring anthropometric parameters included fetal, neonatal, and childhood weight and body composition data. Thirty-three studies (n = 4,944), from geographical locations including Europe, North Africa, the Middle East, Asia, Australia/New Zealand, and the United States/Latin America, met eligibility criteria. Twenty-two studies (n = 2,801) randomised women to metformin versus insulin, 8 studies (n = 1,722) to glyburide versus insulin, and 3 studies (n = 421) to metformin versus glyburide. Eleven studies (n = 2,204) reported maternal outcomes. No differences in fasting blood glucose (FBS), random blood glucose (RBS), or glycated haemoglobin (HbA1c) were reported. No studies reported fetal growth parameters. Thirty-three studies (n = 4,733) reported birth weight. Glyburide-exposed neonates were heavier at birth (58.20 g, 95% confidence interval [CI] 10.10-106.31, p = 0.02) with increased risk of macrosomia (odds ratio [OR] 1.38, 95% CI 1.01-1.89, p = 0.04) versus neonates of insulin-treated mothers. Metformin-exposed neonates were born lighter (-73.92 g, 95% CI -114.79 to -33.06 g, p < 0.001) with reduced risk of macrosomia (OR 0.60, 95% CI 0.45-0.79, p < 0.001) than insulin-exposed neonates. Metformin-exposed neonates were born lighter (-191.73 g, 95% CI -288.01 to -94.74, p < 0.001) with a nonsignificant reduction in macrosomia risk (OR 0.32, 95% CI 0.08-1.19, I2 = 0%, p = 0.09) versus glyburide-exposed neonates. Glyburide-exposed neonates had a nonsignificant increase in total fat mass (103.2 g, 95% CI -3.91 to 210.31, p = 0.06) and increased abdominal (0.90 cm, 95% CI 0.03-1.77, p = 0.04) and chest circumferences (0.80 cm, 95% CI 0.07-1.53, p = 0.03) versus insulin-exposed neonates. Metformin-exposed neonates had decreased ponderal index (-0.13 kg/m3, 95% CI -0.26 to -0.00, p = 0.04) and reduced head (-0.21, 95% CI -0.39 to -0.03, p = 0.03) and chest circumferences (-0.34 cm, 95% CI -0.62 to -0.05, p = 0.02) versus the insulin-treated group. Metformin-exposed neonates had decreased ponderal index (-0.09 kg/m3, 95% CI -0.17 to -0.01, p = 0.03) versus glyburide-exposed neonates. Study limitations include heterogeneity in dosing, heterogeneity in GDM diagnostic criteria, and few studies reporting longitudinal growth outcomes. CONCLUSIONS: Maternal randomisation to glyburide resulted in heavier neonates with a propensity to increased adiposity versus insulin- or metformin-exposed groups. Metformin-exposed neonates were lighter with reduced lean mass versus insulin- or glyburide-exposed groups, independent of maternal glycaemic control. Oral anti-hyperglycaemics cross the placenta, so effects on fetal anthropometry could result from direct actions on the fetus and/or placenta. We highlight a need for further studies examining the effects of intrauterine exposure to antidiabetic agents on longitudinal growth, and the importance of monitoring fetal growth and maternal glycaemic control when treating GDM. This review protocol was registered with PROSPERO (CRD42019134664/CRD42018117503).

A suboptimal maternal diet combined with accelerated postnatal growth results in an altered aging profile in the thymus of male rats.

Reduced fetal nutrition and rapid postnatal growth accelerates the aging phenotype in many organ systems; however, effects on the immune system are unclear. We addressed this by studying the thymus from a rat model of developmental programming. The recuperated group was generated by in utero protein restriction, followed by cross-fostering to control-fed mothers, and were then compared with controls. Fat infiltration and adipocyte size increased with age ( P < 0.001) and in recuperated thymi ( P < 0.05). Cortex/medulla ratio decreased with age ( P < 0.001) and decreased ( P < 0.05) in 12-mo recuperated thymi. Age-associated decreases in thymic-epithelial cell ( P < 0.01) and thymocyte markers ( P < 0.01) were observed in both groups and was decreased ( P < 0.05) in recuperated thymi. These data demonstrate effects of developmental programming upon thymic involution. The recuperated group had longer thymic telomeres than controls ( P < 0.001) at 22 d and at 3 mo, which was associated with increased expression of telomere-length maintenance molecules [telomerase RNA component ( Terc; P < 0.01), P23 ( P = 0.02), and Ku70 and Ku80 ( P < 0.01)]. By 12 mo, recuperated offspring had shorter thymic telomeres than controls had ( P < 0.001) and reduced DNA damage-response markers [( DNA-PKcs, Mre11 ( P < 0.01), Xrcc4 ( P = 0.02), and γ-H2ax ( P < 0.001], suggesting failure of earlier compensatory responses. Our results suggest that low birth weight with rapid postnatal growth results in premature thymic maturation, resulting in accelerated thymic aging. This could lead to increased age-associated vulnerability to infection.-Tarry-Adkins, J. L., Aiken, C. E., Ashmore, T. J., Fernandez-Twinn, D. S., Chen, J.-H., Ozanne, S. E. A suboptimal maternal diet combined with accelerated postnatal growth results in an altered aging profile in the thymus of male rats.

Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats.

Chronic fetal hypoxia is a common complication observed in human pregnancy, impacting pregnancies across global contexts. Exposure to chronic intrauterine hypoxia has major short- and long-term consequences for offspring health. However, the impact of chronic gestational hypoxia on female reproductive system development is unknown. We aimed to understand the impact of exposure to chronic fetal hypoxia on the developing female reproductive system. Wistar rat dams underwent normoxia (21%) or hypoxia (13%) during pregnancy. Postnatally, all female offspring were maintained in normoxic conditions into early adulthood. Female rats exposed to chronic gestational hypoxia (13%) during their intrauterine development had decreased ovarian primordial follicular reserve compared to controls (P < 0.05). Adult females who had been exposed to chronic fetal hypoxia had significantly reduced somatic ovarian telomere length (P < 0.05) and reduced ovarian protein expression of KU70, a critical component of the DNA-activated protein kinase repair complex (P < 0.01). Gene expression of NADPH oxidase 2-mediated oxidative stress markers was increased (P < 0.05). Exposure to chronic hypoxia during fetal development leads to accelerated aging of the somatic ovary and decreased ovarian reserve in adulthood. Ovarian aging is highly sensitive to gestational hypoxia, with implications for future fertility in next-generation offspring of high-risk pregnancies.-Aiken, C. E., Tarry-Adkins, J. L., Spiroski, A.-M., Nuzzo, A. M., Ashmore, T. J., Rolfo, A., Sutherland, M. J., Camm, E. J., Giussani, D. A., Ozanne, S. E. Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats.

Poor Early Growth and Age-Associated Disease.

The prevalence of age-associated disease is increasing at a striking rate globally and there is evidence to suggest that the ageing process may actually begin before birth. It has been well-established that the status of both the maternal and early postnatal environments into which an individual is exposed can have huge implications for the risk of developing age-associated disease, including cardiovascular disease (CVD), type-2 diabetes (T2D) and obesity in later life. Therefore, the dissection of underlying molecular mechanisms to explain this phenomenon, known as 'developmental programming' is a highly investigated area of research. This book chapter will examine the epidemiological evidence and the animal models of suboptimal maternal and early postnatal environments and will discuss the progress being made in the development of safe and effective intervention strategies which ultimately could target those 'programmed' individuals who are known to be at-risk of age-associated disease.

Chronic fetal hypoxia disrupts the peri-conceptual environment in next-generation adult female rats.

KEY POINTS: Exposure to chronic hypoxia during gestation influences long-term health and development, including reproductive capacity, across generations. If the peri-conceptual environment in the developing oviduct is affected by gestational hypoxia, then this could have implications for later fertility and the health of future generations. In the present study, we show that the oviducts of female rats exposed to chronic hypoxia in utero have reduced telomere length, decreased mitochondrial DNA biogenesis and increased oxidative stress The results of the present study show that exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in early adulthood and they help us understand how exposure to hypoxia during development could influence reproductive health across generations. ABSTRACT: Exposure to chronic hypoxia during fetal development has important effects on immediate and long-term outcomes in offspring. Adverse impacts in adult offspring include impairment of cardiovascular function, metabolic derangement and accelerated ovarian ageing. However, it is not known whether other aspects of the female reproductive system may be similarly affected. In the present study, we examined the impact of chronic gestational hypoxia on the developing oviduct. Wistar rat dams were randomized to either normoxia (21%) or hypoxia (13%) from day 6 post-mating until delivery. Post-delivery female offspring were maintained in normoxia until 4 months of age. Oviductal gene expression was assayed at the RNA (quantitative RT-PCR) and protein (western blotting) levels. Oviductal telomere length was assayed using Southern blotting. Oviductal telomere length was reduced in the gestational hypoxia-exposed animals compared to normoxic controls (P < 0.01). This was associated with a specific post-transcriptional reduction in the KU70 subunit of DNA-pk in the gestational hypoxia-exposed group (P < 0.05). Gestational hypoxia-exposed oviducts also showed evidence of decreased mitochondrial DNA biogenesis, reduced mtDNA copy number (P < 0.05) and reduced gene expression of Tfam (P < 0.05) and Pgc1α (P < 0.05). In the hypoxia-exposed oviducts, there was upregulation of mitochondrial-specific anti-oxidant defence enzymes (MnSOD; P < 0.01). Exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in adulthood. The oviduct plays a central role in early development as the site of gamete transport, syngamy, and early development; hence, accelerated ageing of the oviductal environment could have important implications for fertility and the health of future generations.

Neonatal, infant, and childhood growth following metformin versus insulin treatment for gestational diabetes: A systematic review and meta-analysis.

BACKGROUND: Metformin is increasingly offered as an acceptable and economic alternative to insulin for treatment of gestational diabetes mellitus (GDM) in many countries. However, the impact of maternal metformin treatment on the trajectory of fetal, infant, and childhood growth is unknown. METHODS AND FINDINGS: PubMed, Ovid Embase, Medline, Web of Science, ClinicalTrials.gov, and the Cochrane database were systematically searched (from database inception to 26 February 2019). Outcomes of GDM-affected pregnancies randomised to treatment with metformin versus insulin were included (randomised controlled trials and prospective randomised controlled studies) from cohorts including European, American, Asian, Australian, and African women. Studies including pregnant women with pre-existing diabetes or non-diabetic women were excluded, as were trials comparing metformin treatment with oral glucose-lowering agents other than insulin. Two reviewers independently assessed articles for eligibility and risk of bias, and conflicts were resolved by a third reviewer. Outcome measures were parameters of fetal, infant, and childhood growth, including weight, height, BMI, and body composition. In total, 28 studies (n = 3,976 participants) met eligibility criteria and were included in the meta-analysis. No studies reported fetal growth parameters; 19 studies (n = 3,723 neonates) reported measures of neonatal growth. Neonates born to metformin-treated mothers had lower birth weights (mean difference -107.7 g, 95% CI -182.3 to -32.7, I2 = 83%, p = 0.005) and lower ponderal indices (mean difference -0.13 kg/m3, 95% CI -0.26 to 0.00, I2 = 0%, p = 0.04) than neonates of insulin-treated mothers. The odds of macrosomia (odds ratio [OR] 0.59, 95% CI 0.46 to 0.77, p < 0.001) and large for gestational age (OR 0.78, 95% CI 0.62 to 0.99, p = 0.04) were lower following maternal treatment with metformin compared to insulin. There was no difference in neonatal height or incidence of small for gestational age between groups. Two studies (n = 411 infants) reported measures of infant growth (18-24 months of age). In contrast to the neonatal phase, metformin-exposed infants were significantly heavier than those in the insulin-exposed group (mean difference 440 g, 95% CI 50 to 830, I2 = 4%, p = 0.03). Three studies (n = 520 children) reported mid-childhood growth parameters (5-9 years). In mid-childhood, BMI was significantly higher (mean difference 0.78 kg/m2, 95% CI 0.23 to 1.33, I2 = 7%, p = 0.005) following metformin exposure than following insulin exposure, although the difference in absolute weights between the groups was not significantly different (p = 0.09). Limited evidence (1 study with data treated as 2 cohorts) suggested that adiposity indices (abdominal [p = 0.02] and visceral [p = 0.03] fat volumes) may be higher in children born to metformin-treated compared to insulin-treated mothers. Study limitations include heterogeneity in metformin dosing, heterogeneity in diagnostic criteria for GDM, and the scarcity of reporting of childhood outcomes. CONCLUSIONS: Following intrauterine exposure to metformin for treatment of maternal GDM, neonates are significantly smaller than neonates whose mothers were treated with insulin during pregnancy. Despite lower average birth weight, metformin-exposed children appear to experience accelerated postnatal growth, resulting in heavier infants and higher BMI by mid-childhood compared to children whose mothers were treated with insulin. Such patterns of low birth weight and postnatal catch-up growth have been reported to be associated with adverse long-term cardio-metabolic outcomes. This suggests a need for further studies examining longitudinal perinatal and childhood outcomes following intrauterine metformin exposure. This review protocol was registered with PROSPERO under registration number CRD42018117503.

Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet.

Maternal diet during pregnancy influences the later life reproductive potential of female offspring. We investigate the molecular mechanisms underlying the depletion of ovarian follicular reserve in young adult females following exposure to obesogenic diet in early life. Furthermore, we explore the interaction between adverse maternal diet and postweaning diet in generating reduced ovarian reserve. Female mice were exposed to either maternal obesogenic (high fat/high sugar) or maternal control dietin uteroand during lactation, then weaned onto either obesogenic or control diet. At 12 wk of age, the offspring ovarian reserve was depleted following exposure to maternal obesogenic diet (P< 0.05), but not postweaning obesogenic diet. Maternal obesogenic diet was associated with increased mitochondrial DNA biogenesis (copy numberP< 0.05; transcription factor A, mitochondrial expressionP< 0.05), increased mitochondrial antioxidant defenses [manganese superoxide dismutase (MnSOD)P< 0.05; copper/zinc superoxide dismutaseP< 0.05; glutathione peroxidase 4P< 0.01] and increased lipoxygenase expression (arachidonate 12-lipoxygenaseP< 0.05; arachidonate 15-lipoxygenaseP< 0.05) in the ovary. There was also significantly increased expression of the transcriptional regulator NF-κB (P< 0.05). There was no effect of postweaning diet on any measured ovarian parameters. Maternal diet thus plays a central role in determining follicular reserve in adult female offspring. Our observations suggest that lipid peroxidation and mitochondrial biogenesis are the key intracellular pathways involved in programming of ovarian reserve.-Aiken, C. E., Tarry-Adkins, J. L., Penfold, N. C., Dearden, L., Ozanne, S. E. Decreased ovarian reserve, dysregulation of mitochondrial biogenesis, and increased lipid peroxidation in female mouse offspring exposed to an obesogenic maternal diet.

Divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease.

Aging and developmental programming are both associated with oxidative stress and endothelial dysfunction, suggesting common mechanistic origins. However, their interrelationship has been little explored. In a rodent model of programmed cardiovascular dysfunction we determined endothelial function and vascular telomere length in young (4 mo) and aged (15 mo) adult offspring of normoxic or hypoxic pregnancy with or without maternal antioxidant treatment. We show loss of endothelial function [maximal arterial relaxation to acetylcholine (71 ± 3 vs. 55 ± 3%) and increased vascular short telomere abundance (4.2-1.3 kb) 43.0 ± 1.5 vs. 55.1 ± 3.8%) in aged vs. young offspring of normoxic pregnancy (P < 0.05). Hypoxic pregnancy in young offspring accelerated endothelial dysfunction (maximal arterial relaxation to acetylcholine: 42 ± 1%, P < 0.05) but this was dissociated from increased vascular short telomere length abundance. Maternal allopurinol rescued maximal arterial relaxation to acetylcholine in aged offspring of normoxic or hypoxic pregnancy but not in young offspring of hypoxic pregnancy. Aged offspring of hypoxic allopurinol pregnancy compared with aged offspring of untreated hypoxic pregnancy had lower levels of short telomeres (vascular short telomere length abundance 35.1 ± 2.5 vs. 48.2 ± 2.6%) and of plasma proinflammatory chemokine (24.6 ± 2.8 vs. 36.8 ± 5.5 pg/ml, P < 0.05). These data provide evidence for divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease, and aging being decelerated by antioxidants even prior to birth.-Allison, B. J., Kaandorp, J. J., Kane, A. D., Camm, E. J., Lusby, C., Cross, C. M., Nevin-Dolan, R., Thakor, A. S., Derks, J. B., Tarry-Adkins, J. L., Ozanne, S. E., Giussani, D. A. Divergence of mechanistic pathways mediating cardiovascular aging and developmental programming of cardiovascular disease.

Ageing is associated with molecular signatures of inflammation and type 2 diabetes in rat pancreatic islets.

AIMS/HYPOTHESIS: Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas. METHODS: Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods. RESULTS: Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1). CONCLUSIONS/INTERPRETATION: Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.

Transgenerational effects of maternal diet on metabolic and reproductive ageing.

The early-life environment, in particular maternal diet during pregnancy, influences a wide range of organs and systems in adult offspring. Mounting evidence suggests that developmental programming can also influence health and disease in grand-offspring. Transgenerational effects can be defined as those persisting into an F2 generation, where the F0 mother experiences suboptimal diet during her pregnancy. In this review, we critically examine evidence for transgenerational developmental programming effects in human populations, focusing on metabolic and reproductive outcomes. We discuss evidence from historical cohorts suggesting that grandchildren of women exposed to famine and other dietary alterations during pregnancy may experience increased rates of later health complications than their control counterparts. The methodological difficulties with transgenerational studies in human cohorts are explored. In particular, the problems with assessing reproductive outcomes in human populations are discussed. In light of the relative paucity of evidence available from human cohorts, we consider key insights from transgenerational experimental animal models of developmental programming by maternal diet; data are drawn from a range of rodent models, as well as the guinea-pig and the sheep. The evidence for different potential mechanisms of transgenerational inheritance or re-propagation of developmental programming effects is evaluated. Transgenerational effects could be transmitted through methylation of the gametes via the paternal and maternal lineage, as well as other possible mechanisms via the maternal lineage. Finally, future directions for exploring these underlying mechanisms further are proposed, including utilizing large, well-characterized, prospective pregnancy cohorts that include biobanks, which have been established in various populations during the last few decades.

Nutritional programming of coenzyme Q: potential for prevention and intervention?

Low birth weight and rapid postnatal growth increases risk of cardiovascular-disease (CVD); however, underlying mechanisms are poorly understood. Previously, we demonstrated that rats exposed to a low-protein diet in utero that underwent postnatal catch-up growth (recuperated) have a programmed deficit in cardiac coenzyme Q (CoQ) that was associated with accelerated cardiac aging. It is unknown whether this deficit occurs in all tissues, including those that are clinically accessible. We investigated whether aortic and white blood cell (WBC) CoQ is programmed by suboptimal early nutrition and whether postweaning dietary supplementation with CoQ could prevent programmed accelerated aging. Recuperated male rats had reduced aortic CoQ [22 d (35±8.4%; P<0.05); 12 m (53±8.8%; P<0.05)], accelerated aortic telomere shortening (P<0.01), increased DNA damage (79±13% increase in nei-endonucleaseVIII-like-1), increased oxidative stress (458±67% increase in NAPDH-oxidase-4; P<0.001), and decreased mitochondrial complex II-III activity (P<0.05). Postweaning dietary supplementation with CoQ prevented these detrimental programming effects. Recuperated WBCs also had reduced CoQ (74±5.8%; P<0.05). Notably, WBC CoQ levels correlated with aortic telomere-length (P<0.0001) suggesting its potential as a diagnostic marker of vascular aging. We conclude that early intervention with CoQ in at-risk individuals may be a cost-effective and safe way of reducing the global burden of CVDs.

Suboptimal nutrition in utero causes DNA damage and accelerated aging of the female reproductive tract.

Early life exposure to adverse environments can lead to a variety of metabolic and cardiovascular diseases in offspring. We hypothesize that female reproductive function may also be affected, with subsequent implications for fertility. We used an established maternal low-protein model where animals are born small but undergo rapid postnatal catch-up growth by suckling a control-fed dam (recuperated offspring). Markers of oxidative stress and cellular aging in reproductive tract tissues were assessed at 3 and 6 mo of age. Recuperated offspring had lower birth weight than controls (P<0.01) but caught up during lactation. 4-Hydroxynonenal (4HNE; an indicator of oxidative stress) was increased in recuperated animals compared with controls in both ovaries and oviducts at 6 mo. At 3 and 6 mo, ovaries and oviducts of recuperated offspring had increased mitochondrial (mt)DNA copy number (P<0.01). By contrast, germ-line cells showed no difference in mtDNA copy number, suggesting they were protected from suboptimal maternal nutrition. Oviduct and somatic ovarian telomere length declined more rapidly with age in recuperated animals. This accelerated cellular aging was associated with a declined ovarian reserve in developmentally programmed animals. These findings have significant clinical implications in light of worldwide trends to delayed childbearing.

Poor maternal nutrition followed by accelerated postnatal growth leads to alterations in DNA damage and repair, oxidative and nitrosative stress, and oxidative defense capacity in rat heart.

Low birth weight and accelerated postnatal growth lead to increased risk of cardiovascular disease. We reported previously that rats exposed to a low-protein diet in utero and postnatal catch-up growth (recuperated) develop metabolic dysfunction and have reduced life span. Here we explored the hypothesis that cardiac oxidative and nitrosative stress leading to DNA damage and accelerated cellular aging could contribute to these phenotypes. Recuperated animals had a low birth weight (P<0.001) but caught up in weight to controls during lactation. At weaning, recuperated cardiac tissue had increased (P<0.05) protein nitrotyrosination and DNA single-stranded breaks. This condition was preceded by increased expression of DNA damage repair molecules 8-oxoguanine-DNA-glycosylase-1, nei-endonuclease-VIII-like, X-ray-repair-complementing-defective-repair-1, and Nthl endonuclease III-like-1 on d 3. These differences were maintained on d 22 and became more pronounced in the case of 8-oxoguanine-DNA-glycosylase-1 and nei-endonuclease-VIII-like. This was accompanied by increases in xanthine oxidase (P<0.001) and NADPH oxidase (P<0.05), major sources of reactive oxygen species (ROS). The detrimental effects of increased ROS in recuperated offspring may be exaggerated at 22 d by reductions (P<0.001) in the antioxidant enzymes peroxiredoxin-3 and CuZn-superoxide-dismutase. We conclude that poor fetal nutrition followed by accelerated postnatal growth results in increased cardiac nitrosative and oxidative-stress and DNA damage, which could contribute to age-associated disease risk.

The Uterine Environment and Childhood Obesity Risk: Mechanisms and Predictions.

PURPOSE OF REVIEW: Childhood obesity is a growing health problem in many populations, hence the urgent need to unravel the underlying mechanisms. Some evidence suggests that exposure to suboptimal intrauterine environments can program foetal metabolic health, with adverse consequences in later life, including susceptibility to childhood obesity. FINDINGS: Factors such as high and low foetal birth weight, excessive gestational-weight-gain, maternal stress and smoking are all associated with increased risk of childhood obesity in observational studies. Animal models, where both genetic background and the postnatal environment can be carefully controlled, suggest that several different mechanisms, including epigenetic changes, dysregulation of adipose tissue development and programming of appetite, may be key drivers of developmental programming of childhood obesity. However, the influence of genetics and the post-natal environment are much more difficult to disentangle as independent effects in human studies, which are also complicated by low follow-up rates. Suboptimal intrauterine environments interact with maternal and foetal genetics and with the postnatal environment to contribute to the risk of childhood obesity. Maternal metabolic challenges, for example obesity and insulin resistance, contribute to the risk of foetal overgrowth and subsequent adiposity in childhood. To protect the long-term health of populations, research focusing on effective means of identifying and intervening in the transgenerational cycle of childhood obesity is required.

Impact of Metformin Treatment on Human Placental Energy Production and Oxidative Stress.

Metformin is increasingly prescribed in pregnancy, with beneficial maternal effects. However, it is not known how metformin-treatment impacts metabolism and energy production in the developing feto-placental unit. We assessed the human placental response to metformin using both in vivo and in vitro treated samples. trophoblasts were derived from placentas collected from non-laboured Caesarean deliveries at term, then treated in vitro with metformin (0.01 mM, 0.1 mM or vehicle). Metformin-concentrations were measured using liquid-chromatography mass-spectrometry. Oxygen consumption in cultured-trophoblasts was measured using a Seahorse-XF Mito Stress Test. Markers of oxidative-stress were assayed using qRT-PCR. Metformin-transporter mRNA and protein-levels were determined by quantitative RT-PCR and Western-blotting respectively. Metformin concentrations were also measured in sample trios (maternal plasma/fetal plasma/placental tissue) from pregnancies exposed to metformin on clinical-grounds. Maternal and fetal metformin concentrations in vivo were highly correlated over a range of concentrations (R2 = 0.76, p < 0.001; average fetal:maternal ratio 1.5; range 0.8-2.1). Basal respiration in trophoblasts was reduced by metformin treatment (0.01 mM metformin; p < 0.05, 0.1 mM metformin; p < 0.001). Mitochondrial-dependent ATP production and proton leak were reduced after treatment with metformin (p < 0.001). Oxidative stress markers were significantly reduced in primary-trophoblast-cultures following treatment with metformin. There is a close linear relationship between placental, fetal, and maternal metformin concentrations. Primary-trophoblast cultures exposed to clinically-relevant metformin concentrations have reduced mitochondrial-respiration, mitochondrial-dependent ATP-production, and reduced markers of oxidative-stress. Given the crucial role of placental energy-production in supporting fetal growth and well-being during pregnancy, the implications of these findings are concerning for intrauterine fetal growth and longer-term metabolic programming in metformin-exposed pregnancies.

Poor maternal nutrition leads to alterations in oxidative stress, antioxidant defense capacity, and markers of fibrosis in rat islets: potential underlying mechanisms for development of the diabetic phenotype in later life.

Low birth weight is associated with glucose intolerance, insulin resistance, and type 2 diabetes (T2D) in later life. Good evidence indicates that the environment plays an important role in this relationship. However, the mechanisms underlying these relationships are defined poorly. Islets are particularly susceptible to oxidative stress, and this condition combined with fibrosis is thought to be instrumental in T2D pathogenesis. Here we use our maternal low-protein (LP) rat model to determine the effect of early diet on oxidative stress and fibrosis in pancreatic islets of male offspring at 3 and 15 mo of age. Islet xanthine oxidase (XO) expression was increased in 15-mo LP offspring, which suggests increased oxidative-stress. Manganese superoxide-dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), and heme oxygenase-1 (HO-1) (antioxidant enzymes) were reduced significantly in LP offspring, which indicated impairment of oxidative defense. Expression of fibrosis markers collagen I and collagen III also increased in 15-mo LP offspring. Angiotensin II receptor type I (AT(II)R(1)), induced by hyperglycemia and oxidative-stress, was significantly up-regulated in 15-mo LP offspring. Lipid peroxidation was also increased in 15-mo LP animals. We conclude that maternal protein restriction causes age-associated increased oxidative stress, impairment of oxidative defense, and fibrosis. These findings provide mechanisms by which suboptimal early nutrition can lead to T2D development later in life.