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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., Medical Research Council New Investigator Grant (MR/T016701/1) NIHR Cambridge Biomedical Research Centre (146281) Medical Research Council (MC_UU_00014/4) (MR/R014167/1) British Heart Foundation (RG/17/12/33167) (PG/20/11/34957) (RE/18/5/34216) Next Generation Fellowship from the Centre for Trophoblast Research

Funder: Isaac Newton Trust/Wellcome Trust ISSF/ University of Cambridge Joint Research Grant, 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).

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.

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

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.

Developmental programming phenotypes can be recapitulated in subsequent generations not directly exposed to the initial suboptimal intrauterine environment. A maternal low-protein diet during pregnancy and postnatal catch-up growth (‘recuperated’) alters insulin signaling and inflammation in rat offspring (F1-generation). We aimed to establish if this phenotype is also present in F2-generation females. Insulin-receptor-substrate-1 protein expression was decreased in para-ovarian adipose tissue at 3 months in offspring exposed to a grand-maternal low-protein diet (F2-recuperated), vs. F2-control animals (p

‘Developmental programming’, which occurs as a consequence of suboptimal in utero and early environments, can be associated with metabolic dysfunction in later life, including an increased incidence of cardiovascular disease and type 2 diabetes, and predisposition of older men to sarcopenia. However, the molecular mechanisms underpinning these associations are poorly understood. Many conditions associated with developmental programming are also known to be associated with the aging process. We therefore utilized our well-established rat model of low birth weight and accelerated postnatal catch-up growth (termed ‘recuperated’) in this study to establish the effects of suboptimal maternal nutrition on age-associated factors in skeletal muscle. We demonstrated accelerated telomere shortening (a robust marker of cellular aging) as evidenced by a reduced frequency of long telomeres (48.5-8.6 kb) and an increased frequency of short telomeres (4.2-1.3 kb) in vastus lateralis muscle from aged recuperated offspring compared to controls. This was associated with increased protein expression of the DNA-damage-repair marker 8-oxoguanine-glycosylase (OGG1) in recuperated offspring. Recuperated animals also demonstrated an oxidative stress phenotype, with decreased citrate synthase activity, increased electron-transport-complex activities of complex I, complex II-III and complex IV (all markers of functional mitochondria), and increased xanthine oxidase (XO), p67phox and nuclear-factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Recuperated offspring also demonstrated increased antioxidant defense capacity, with increased protein expression of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase and heme oxygenase-1 (HO1), all of which are known targets of NF-κB and can be upregulated as a consequence of oxidative stress. Recuperated offspring also had a pro-inflammatory phenotype, as evidenced by increased tumor necrosis factor-α (TNFα) and interleukin-1β (IL1β) protein levels. Taken together, we demonstrate, for the first time to our knowledge, an accelerated aging phenotype in skeletal muscle in the context of developmental programming. These findings may pave the way for suitable interventions in at-risk populations., Summary: Muscle of ‘developmentally programmed’ rat offspring demonstrated accelerated aging and oxidative stress, which could explain why some individuals are at greater risk of developing age-associated muscular dysfunction than others.

Background: It is well established that low birth weight and accelerated postnatal growth increase the risk of liver dysfunction in later life. However, molecular mechanisms underlying such developmental programming are not well characterized, and potential intervention strategies are poorly defined. Objectives: We tested the hypotheses that poor maternal nutrition and accelerated postnatal growth would lead to increased hepatic fibrosis (a pathological marker of liver dysfunction) and that postnatal supplementation with the antioxidant coenzyme Q10 (CoQ10) would prevent this programmed phenotype. Design: A rat model of maternal protein restriction was used to generate low-birth-weight offspring that underwent accelerated postnatal growth (termed “recuperated”). These were compared with control rats. Offspring were weaned onto standard feed pellets with or without dietary CoQ10 (1 mg/kg body weight per day) supplementation. At 12 mo, hepatic fibrosis, indexes of inflammation, oxidative stress, and insulin signaling were measured by histology, Western blot, ELISA, and reverse transcriptase–polymerase chain reaction. Results: Hepatic collagen deposition (diameter of deposit) was greater in recuperated offspring (mean ± SEM: 12 ± 2 μm) than in controls (5 ± 0.5 μm) (P < 0.001). This was associated with greater inflammation (interleukin 6: 38% ± 24% increase; P < 0.05; tumor necrosis factor α: 64% ± 24% increase; P < 0.05), lipid peroxidation (4-hydroxynonenal, measured by ELISA: 0.30 ± 0.02 compared with 0.19 ± 0.05 μg/mL per μg protein; P < 0.05), and hyperinsulinemia (P < 0.05). CoQ10 supplementation increased (P < 0.01) hepatic CoQ10 concentrations and ameliorated liver fibrosis (P < 0.001), inflammation (P < 0.001), some measures of oxidative stress (P < 0.001), and hyperinsulinemia (P < 0.01). Conclusions: Suboptimal in utero nutrition combined with accelerated postnatal catch-up growth caused more hepatic fibrosis in adulthood, which was associated with higher indexes of oxidative stress and inflammation and hyperinsulinemia. CoQ10 supplementation prevented liver fibrosis accompanied by downregulation of oxidative stress, inflammation, and hyperinsulinemia.

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.

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.

Obese pregnancies are not only associated with adverse consequences for the mother but also the long-term health of her child. Human studies have shown that individuals from obese mothers are at increased risk of premature death from cardiovascular disease (CVD), but are not able to define causality. This study aimed to determine causality using a mouse model of maternal diet-induced obesity. Obesity was induced in female C57BL/6 mice by feeding a diet rich in simple sugars and saturated fat 6 weeks prior to pregnancy and throughout pregnancy and lactation. Control femaleswere fed laboratory chow. Male offspring from both groups were weaned onto chow and studied at 3, 5, 8 and 12 weeks of age for gross cardiac morphometry using stereology, cardiomyocyte cell area by histology and cardiac fetal gene expression using qRT-PCR. Cardiac function was assessed by isolated Langendorff technology at 12 weeks of age and hearts were analysed at the protein level for the expression of the β1 adrenergic receptor, muscarinic type-2 acetylcholine receptor and proteins involved in cardiac contraction. Offspring from obese mothers develop pathologic cardiac hypertrophy associated with re-expression of cardiac fetal genes. By young adulthood these offspring developed severe systolic and diastolic dysfunction and cardiac sympathetic dominance. Importantly, cardiac dysfunction occurred in the absence of any change in corresponding body weight and despite the offspring eating a healthy low fat diet. These findings provide a causal link to explain human observations relating maternal obesity with premature death from CVD in her offspring.