Dr Joshua Fisher

Background: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. Methods: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30¿kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; = 3 ATPIII report criteria, n = 39). Results: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3¿4¿µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017¿0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. Conclusions: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5¿µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Purpose: To compare physiological responses to submaximal cycling and sprint cycling performance in women using oral contraceptives (WomenOC) and naturally cycling women (WomenNC) and to determine whether N-acetylcysteine (NAC) supplementation mediates these responses. Methods: Twenty recreationally trained women completed five exercise trials (i.e., an incremental cycling test, a familiarisation trial, a baseline performance trial and two double-blind crossover intervention trials). During the intervention trials participants supplemented with NAC or a placebo 1 h before exercise. Cardiopulmonary parameters and blood biochemistry were assessed during 40 min of fixed-intensity cycling at 105% of gas-exchange threshold and after 1-km cycling time-trial. Results: WomenOC had higher ventilation (ß [95% CI] = 0.07 L·min-1 [0.01, 0.14]), malondialdehydes (ß = 12.00¿mmol·L-1 [6.82, 17.17]) and C-reactive protein (1.53¿mg·L-1 [0.76, 2.30]), whereas glutathione peroxidase was lower (ß = 22.62¿mU·mL-1 [-¿41.32, -¿3.91]) compared to WomenNC during fixed-intensity cycling. Plasma thiols were higher at all timepoints after NAC ingestion compared to placebo, irrespective of group (all p < 0.001; d = 1.45 to 2.34). For WomenNC but not WomenOC, the exercise-induced increase in malondialdehyde observed in the placebo trial was blunted after NAC ingestion, with lower values at 40¿min (p = 0.018; d = 0.73). NAC did not affect cycling time-trial performance. Conclusions: Blood biomarkers relating to oxidative stress and inflammation are elevated in WomenOC during exercise. There may be an increased strain on the endogenous antioxidant system during exercise, since NAC supplementation in WomenOC did not dampen the exercise-induced increase in malondialdehyde. Future investigations should explore the impact of elevated oxidative stress on exercise adaptations or recovery from exercise in WomenOC.

Career trajectories in science are often unpredictable, with many early and mid-career researchers working multiple successive fixed-term contracts, and physically relocating to take up employment opportunities. Whilst this can provide exciting opportunities to change research direction, acquire new skills, and see the world, the precarity of this scenario is also a significant cause of anxiety for many, and can have a negative impact on their ability to maintain career momentum and trajectory, access institutional financial benefits, or make long term career or financial plans. Here, we build on a pair of workshops held at the 2021 International Federation of Placenta Associations annual conference to discuss two key areas important to help early career researchers navigate their careers ¿ building an academic profile, and the financial ramifications of academic careers.

Introduction: The human placenta has a defined lifespan and placental aging is a key feature as pregnancy progresses. Placental aging and mitochondrial dysfunction are known to play a key role in pregnancy pathophysiology. Premature aging of the placenta has also been linked with placental dysfunction resulting in poor fetal development and premature birth. Methods: The expression of key mitochondrial-related genes were analysed in a series of publicly available databases then expression changes were validated in placental samples collected from term, pre-term, post-term pregnancies and pregnancies complicated by fetal growth restriction (FGR). Gene and protein expression levels of MFN1, MFN2, TFAM, TOMM20, OPA3 and SIRT4 were measured in placental tissues via qPCR and western blotting. Results: Initial analysis found that key mitochondrial transcripts related to biogenesis, bioenergetics and mitophagy clustered by pregnancy trimester. A refined list of 13 mitochondrial-related genes were investigated in additional external datasets of pregnancy complications. In the new cohort, protein expression of MFN1 was decreased in FGR and MFN2 is decreased in post-term placenta. Analysis of placental tissues revealed that TOMM20 gene and protein expression was altered in FGR and post-term placenta. Discussion: MFN1 and MFN2 play a major role in mitochondrial dynamics, and alterations in these markers have been highlighted in early unexplained miscarriage. TOMM20 is an importer protein that plays a major role in mitophagy and changes have also been identified in age-related diseases. Significant changes in MFN1, MFN2 and TOMM20 indicate that mitochondrial regulators play a critical role in placental aging and placental pathophysiology.

Purpose: To examine the temporal changes in blood oxidative stress biomarkers in recreationally-trained women that were naturally-cycling (WomenNC) or using oral contraceptives (WomenOC) across one month. Methods: Blood samples were acquired at three timepoints of the menstrual cycle (1: early-follicular, 2: late-follicular and 3: mid-luteal) and oral contraceptive packet (1: InactiveOC, 2: Mid-activeOC and 3: Late-activeOC) for determination of estradiol, progesterone, oxidative stress, C-reactive protein (CRP) and other cardiometabolic biomarkers in plasma and serum. Results: There was a Group by Time effect on estradiol (p < 0.001, partial ¿2 = 0.64) and progesterone (p < 0.001, partial ¿2 = 0.77). Malondialdehyde, lipid hydroperoxides and CRP concentrations were higher in WomenOC during Late-activeOC compared to InactiveOC (+ 96%, + 23% and + 104%, respectively, p < 0.05). However, there were no changes in these biomarkers across the menstrual cycle in WomenNC (p > 0.05). At all timepoints (i.e., 1, 2 and 3), WomenOC had elevated lipid hydroperoxides (+ 28, + 48% and + 50%) and CRP (+ 71%, + 117% and + 130%) compared to WomenNC (p < 0.05, partial ¿2 > 0.25). There was no Group by Time effect on non-enzymatic antioxidants or glutathione peroxidase; however, glutathione peroxidase was lower in WomenOC, i.e., main effect of group (p < 0.05, partial ¿2 > 0.20). Conclusion: These findings demonstrate that WomenOC not only have higher oxidative stress and CRP than WomenNC, but also a transient increase across one month of habitual oral contraceptive use. Since changes in oxidative stress and CRP often relate to training stress and recovery, these outcomes may have implications to workload monitoring practices in female athletes.

Introduction: Poor gestational outcomes due to placental insufficiency can have lifelong consequences for mother and child. Objective: There is a need for better methods of diagnosis, and elemental metabolomics may provide a means to determine the risk of gestational disorders. Methods: This study used blood plasma samples collected at 36¿weeks' gestation from women who later developed preeclampsia (n = 38), or small-for-gestational age babies (n = 91), along with matched controls (n = 193). Multi-element analysis was conducted by inductively coupled plasma mass spectrometer (ICP-MS), allowing simultaneous measurement of 28 elements. Results: Women who later developed PE, exhibited significantly increased concentrations of K, Rb and Ba. For SGA pregnancies, there was a significant increase in Cu and a decrease in As concentrations. Despite significant differences in single elements, the elemental profile of groups indicated no clustering of control, PE, or SGA samples. Positive predicative values correctly identified approximately 60% of SGA and 70% of PE samples. Conclusion: This is the first-time elemental metabolomics has been used to predict SGA and PE at 36¿weeks. Though significant changes were identified, routine clinical use may be limited but may contribute to a multi marker test. Future analysis should include other biomarkers, metabolic data or clinical measurements made throughout gestation.

Key points: Mitochondrial dysfunction is known to occur in diabetic phenotypes including type 1 and 2 diabetes mellitus. The incidence of gestational diabetes mellitus (GDM) is increasing and defined as the onset of a diabetic phenotype during pregnancy. The role of placental mitochondria in the aetiology of GDM remains unclear and is an emerging area of research. Differing mitochondrial morphologies within the placenta may influence the pathogenesis of the disorder. This study observed mitochondrial dysfunction in GDM placenta when assessing whole tissue. Upon further investigation into mitochondrial isolates from the cytotrophoblast and syncytiotrophoblast, mitochondrial dysfunction appears exaggerated in syncytiotrophoblast. Assessing mitochondrial populations individually enabled the determination of differences between cell lineages of the placenta and established varying levels of mitochondrial dysfunction in GDM, in some instances establishing significance in pathways previously inconclusive or confounded when assessing whole tissue. This research lays the foundation for future work into mitochondrial dysfunction in the placenta and the role it may play in the aetiology of GDM. Abstract: Mitochondrial dysfunction has been associated with diabetic phenotypes, yet the involvement of placental mitochondria in gestational diabetes mellitus (GDM) remains inconclusive. This is in part complicated by the different mitochondrial subpopulations present in the two major trophoblast cell lineages of the placenta. To better elucidate the role of mitochondria in this pathology, this study examined key aspects of mitochondrial function in placentas from healthy pregnancies and those complicated by GDM in both whole tissue and isolated mitochondria. Mitochondrial content, citrate synthase activity, reactive oxygen species production and gene expression regulating metabolic, hormonal and antioxidant control was examined in placental tissue, before examining functional differences between mitochondrial isolates from cytotrophoblast (Cyto-Mito) and syncytiotrophoblast (Syncytio-Mito). Our study observed evidence of mitochondrial dysfunction across multiple pathways when assessing whole placental tissue from GDM pregnancies compared with healthy controls. Furthermore, by examining isolated mitochondria from the cytotrophoblast and syncytiotrophoblast cell lineages of the placenta we established that although both mitochondrial populations were dysfunctional, they were differentially impacted. These data highlight the need to consider changes in mitochondrial subpopulations at the feto-maternal interface when studying pregnancy pathologies.

A normal pregnancy is essential to establishing a healthy start to life. Complications during have been associated with adverse perinatal outcomes and lifelong health problems. The ability to identify risk factors associated with pregnancy complications early in gestation is vitally important for preventing negative foetal outcomes. Maternal nutrition has been long considered vital to a healthy pregnancy, with micronutrients and trace elements heavily implicated in maternofoetal metabolism. This study proposed the use of elemental metabolomics to study multiple elements at 18¿weeks gestation from blood plasma and urine to construct models that could predict outcomes such as small for gestational age (SGA) (n = 10), low placental weight (n = 18), and preterm birth (n = 13) from control samples (n = 87). Samples collected from the Lyell McEwin Hospital in Adelaide, South Australia, were measured for 27 plasma elements and 37 urine elements by inductively coupled plasma mass spectrometry. Exploratory analysis indicated an average selenium concentration 20¿µg/L lower than established reference ranges across all groups, low zinc in preterm (0.64¿µg/L, reference range 0.66¿1.10¿µg/L), and higher iodine in preterm and SGA gestations (preterm 102¿µg/L, SGA 111¿µg/L, reference range 40¿92¿µg/L). Using random forest algorithms with receiver operating characteristic curves, low placental weight was predicted with 86.7% accuracy using plasma, 78.6% prediction for SGA with urine, and 73.5% determination of preterm pregnancies. This study indicates that elemental metabolomic modelling could provide a means of early detection of at-risk pregnancies allowing for more targeted monitoring of mothers, with potential for early intervention strategies to be developed.