Evolution of Key Oxygen-Sensing Genes Is Associated with Hypoxia Tolerance in Fishes.

Low dissolved oxygen (hypoxia) is recognized as a major threat to aquatic ecosystems worldwide. Because oxygen is paramount for the energy metabolism of animals, understanding the functional and genetic drivers of whole-animal hypoxia tolerance is critical to predicting the impacts of aquatic hypoxia. In this study, we investigate the molecular evolution of key genes involved in the detection of and response to hypoxia in ray-finned fishes: the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) oxygen-sensing system, also known as the EGLN (egg-laying nine)-HIF oxygen-sensing system. We searched fish genomes for HIFA and EGLN genes, discovered new paralogs from both gene families, and analyzed protein-coding sites under positive selection. The physicochemical properties of these positively selected amino acid sites were summarized using linear discriminants for each gene. We employed phylogenetic generalized least squares to assess the relationship between these linear discriminants for each HIFA and EGLN and hypoxia tolerance as reflected by the critical oxygen tension (Pcrit) of the corresponding species. Our results demonstrate that Pcrit in ray-finned fishes correlates with the physicochemical variation of positively selected sites in specific HIFA and EGLN genes. For HIF2A, two linear discriminants captured more than 90% of the physicochemical variation of these sites and explained between 20% and 39% of the variation in Pcrit. Thus, variation in HIF2A among fishes may contribute to their capacity to cope with aquatic hypoxia, similar to its proposed role in conferring tolerance to high-altitude hypoxia in certain lineages of terrestrial vertebrates.

Mitochondrial oxygen tension in critically ill patients receiving red blood cell transfusions: a multicenter observational cohort study.

PURPOSE: Currently, there is no marker of efficacy of red blood cell (RBC) transfusion. This study describes the impact of RBC transfusion on mitochondrial oxygen tension (mitoPO2) and mitochondrial oxygen consumption (mitoVO2) in critically ill patients with anemia. METHODS: Critically ill patients with a hemoglobin concentration < 10 g/dL, for whom a single RBC unit had been ordered, were included. MitoPO2 was measured with the COMET device immediately before RBC transfusion, 0.5 h, 1 h, 3 h, and 24 h after RBC transfusion. MitoVO2 was calculated from dynamic mitoPO2 measurements during cessation of local oxygen supply. RESULTS: Sixty-three patients participated, median age 64.0 (interquartile range (IQR) 52.3-72.8) years, median hemoglobin concentration before transfusion 7.4 (IQR 7.1-7.7) g/dL. Median mitoPO2 values were 55.0 (IQR 49.6-63.0) mmHg before RBC transfusion, 51.0 (IQR 41.5-61.2) directly after and 67.3 (IQR 41.6-83.7) at 24 h after RBC transfusion. Median mitoVO2 values were 3.3 (IQR 2.1-5.9) mmHg/s before RBC transfusion, 3.7 (IQR 2.0-5.1) mmHg/s directly after, and 3.1 (IQR 2.5-4.8) mmHg/s 24 h after RBC transfusion. In the higher Hb concentration group (> 7 g/dL), we saw a dissociation of the effect of RBC transfusion on mitoPO2 versus on mitoVO2 values. MitoPO2 and mitoVO2 values were not associated with commonly used parameters of tissue perfusion and oxygenation. CONCLUSION: RBC transfusion did not alter mitoPO2 and mitoVO2 in critically ill patients with anemia. MitoPO2 and mitoVO2 values were not notably associated with Hb concentrations, parameters of severity of illness and markers of tissue perfusion or oxygenation. Given the high baseline value, it cannot be excluded nor confirmed whether RBC can improve low mitoPO2. Trial registration number NCT03092297 (registered 27 March 2017).

Transcutaneous oxygen tension-guided hyperbaric oxygen therapy for preventing skin necrosis after hyaluronic acid filler injections.

Introduction: Hyaluronic acid (HA) fillers, popular for facial cosmetic enhancements, pose risks of vascular complications like skin necrosis due to arterial blockage, necessitating effective treatments such as hyperbaric oxygen therapy (HBOT). Methodology: This study presents a series of cases where measurements of transcutaneous oxygen pressure (TcPO2) informed the application of HBOT for skin necrosis induced by HA. Clinical presentation and outcomes: In cases 1 and 3, following the injection of HA, potential skin necrosis was observed. In addition to standard treatment, TcPO2 revealed values below 40 mmHg, indicating tissue hypoxia. Treatment with HBOT increased TcPO2 levels to above 200 mmHg, suggesting that HBOT could correct the hypoxia. Monitoring TcPO2 levels also aided in determining the optimal time to discontinue HBOT. In cases 2 and 4, patients received standard treatment, resulting in TcPO2 levels above 40 mmHg, indicating adequate tissue oxygenation, and no additional HBOT was administered. All four patients mentioned above showed good clinical recovery. Conclusion: This study investigates the application of TcPO2 measurement technology in aiding decisions on whether to utilize HBOT in the treatment of complications arising from HA fillers, as well as in optimizing HBOT protocols.

The role of DNA damage response in human embryonic stem cells exposed to atmospheric oxygen tension: Implications for embryo development and differentiation.

Previous retrospective cohort studies have found that, compared with oxygen tension in the uterus and fallopian tubes (2 %-8 %), exposure of pre-implantation embryos to atmospheric oxygen tension (AtmO2, 20 %) during assisted reproductive technology(ART) can affect embryo quality, pregnancy outcomes and offspring health. However, current research on the effects and mechanisms of AtmO2 on the development of embryos and offspring is mainly limited to animal experiments. Human embryonic stem cells (hESCs) play a special and irreplaceable role in the study of early human embryonic development. In this study, we used hESCs as a model to elucidate the possible effects and mechanisms of AtmO2 exposure on human embryonic development. We found that exposure to AtmO2 can reduce cell viability, produce oxidative stress, increase DNA damage, initiate DNA repair, activate autophagy, and increase cell apoptosis. We also noticed that approximately 50 % of hESCs survived, adapted and proliferated through high expression of self-renewal and pluripotency regulatory factors, and affected embryoid body differentiation. These data indicate that hESCs experience oxidative stress, accumulation of DNA damage, and activate DNA damage response under the selective pressure of AtmO2.Some hESCs undergo cell death, whereas other hESCs adapt and proliferate through increased expression of self-renewal genes. The current findings provide in vitro evidence that exposure to AtmO2 during the early preimplantation stage negatively affects hESCs.

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro.

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.

Dynamics of transcription is affected by oxygen tension and developmental speed during in vitro production of bovine embryos.

This study examines the impact of oxygen tension and embryo kinetics on gene transcription dynamics in pathways crucial for embryonic preimplantation development, including lipid metabolism, carbohydrate transport and metabolism, mitochondrial function, stress response, apoptosis and transcription regulation. Bovine embryos were generated in vitro and allocated into two groups based on oxygen tension (20% or 5%) at 18 h post insemination (hpi). At 40 hpi, embryos were categorized into Fast (≥4 cells) or Slow (2 cells) groups, resulting in four experimental groups: FCL20, FCL5, SCL20 and SCL5. Embryo collection also occurred at 72 hpi (16-cell stage; groups FMO20, FMO5, SMO20 and SMO5) and at 168 hpi (expanded blastocyst (BL) stage; groups FBL20, FBL5, SBL20 and SBL5). Pools of three embryos per group were analysed in four replicates using inventoried TaqMan assays specific for Bos taurus, targeting 93 genes. Gene expression patterns were analysed using the K-means algorithm, revealing three main clusters: genes with low relative abundance at the cleavage (CL) and 16-cell morula (MO) stages but increased at the BL stage (cluster 1); genes with higher abundances at CL but decreasing at MO and BL (cluster 2); and genes with low levels at CL, higher levels at MO and decreased levels at BL (cluster 3). Within each cluster, genes related to epigenetic mechanisms, cell differentiation events and glucose metabolism were particularly influenced by differences in developmental kinetics and oxygen tension. Fast-developing embryos, particularly those cultured under low oxygen tension, exhibited transcript dynamics more closely resembling that reported in vivo-produced embryos.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Relatedness of hypoxia and hyperthermia tolerances in the Nile tilapia (Oreochromis niloticus) and their relationships with cardiac and gill traits.

In fish, thermal and hypoxia tolerances may be functionally related, as suggested by the oxygen- and capacity-limited thermal tolerance (OCLTT) concept, which explains performance failure at high temperatures due to limitations in oxygen delivery. In this study the interrelatedness of hyperthermia and hypoxia tolerances in the Nile tilapia (Oreochromis niloticus), and their links to cardiorespiratory traits were examined. Different groups of O. niloticus (n = 51) were subjected to hypoxia and hyperthermia challenges and the O2 tension for aquatic surface respiration (ASR pO2) and critical thermal maximum (CTmax) were assessed as measurement endpoints. Gill filament length, total filament number, ventricle mass, length and width were also measured. Tolerance to hypoxia, as evidenced by ASR pO2 thresholds of the individual fish, was highly variable and varied between 0.26 and 3.39 kPa. ASR events increased more profoundly as O2 tensions decreased below 2 kPa. The CTmax values recorded for the O. niloticus individuals ranged from 43.1 to 44.8 °C (Mean: 44.2 ± 0.4 °C). Remarkably, there was a highly significant correlation between ASR pO2 and CTmax in O. niloticus (r = -0.76, p < 0.0001) with ASR pO2 increasing linearly with decreasing CTmax. There were, however, no discernible relationships between the measured cardiorespiratory properties and hypoxia or hyperthermia tolerances. The strong relationship between hypoxia and hyperthermia tolerances in this study may be related to the ability of the cardiorespiratory system to provide oxygen to respiring tissues under thermal stress, and thus provides some support for the OCLTT concept in this species, at least at the level of the entire organism.

Culture of Human Embryos at High and Low Oxygen Levels.

One of the parameters potentially affecting the in vitro growth of preimplantation embryos is the oxygen concentration in the culture environment. An increased oxygen concentration causes the generation of ROS which in turn can cause damage to the cells and seriously disrupt the embryonic development. Previous studies have assessed oxygen concentrations in the fallopian tubes of several mammals of between 5 and 8%, while the oxygen levels in the uterus were found to be even lower; similar measurements have been confirmed in humans. In addition, studies in mammalian embryos showed that low oxygen concentrations improve embryo development. Multiple studies on the effect of the oxygen concentration on human embryos have been conducted so far with diverse methodologies and objectives. Data from these have been included in three meta-analyses. All meta-analyses indicate the potential benefit in favor of a low oxygen concentration, though data are considered to be of a low methodological quality and further studies are considered necessary. However, based on the existing evidence, it is suggested that a low oxygen concentration should be adopted in the routine of the IVF laboratory, especially in the case of blastocyst culture.

Regulation of Oxygen Tension as a Strategy to Control Chondrocytic Phenotype for Cartilage Tissue Engineering and Regeneration.

Cartilage defects and osteoarthritis are health problems which are major burdens on health care systems globally, especially in aging populations. Cartilage is a vulnerable tissue, which generally faces a progressive degenerative process when injured. This makes it the 11th most common cause of global disability. Conservative methods are used to treat the initial phases of the illness, while orthopedic management is the method used for more progressed phases. These include, for instance, arthroscopic shaving, microfracturing and mosaicplasty, and joint replacement as the final treatment. Cell-based implantation methods have also been developed. Despite reports of successful treatments, they often suffer from the non-optimal nature of chondrocyte phenotype in the repair tissue. Thus, improved strategies to control the phenotype of the regenerating cells are needed. Avascular tissue cartilage relies on diffusion for nutrients acquisition and the removal of metabolic waste products. A low oxygen content is also present in cartilage, and the chondrocytes are, in fact, well adapted to it. Therefore, this raises an idea that the regulation of oxygen tension could be a strategy to control the chondrocyte phenotype expression, important in cartilage tissue for regenerative purposes. This narrative review discusses the aspects related to oxygen tension in the metabolism and regulation of articular and growth plate chondrocytes and progenitor cell phenotypes, and the role of some microenvironmental factors as regulators of chondrocytes.

Monitoring of Brain Tissue Oxygen Tension in Cardiac Arrest: a Translational Systematic Review from Experimental to Clinical Evidence.

BACKGROUND: Cardiac arrest (CA) is a sudden event that is often characterized by hypoxic-ischemic brain injury (HIBI), leading to significant mortality and long-term disability. Brain tissue oxygenation (PbtO2) is an invasive tool for monitoring brain oxygen tension, but it is not routinely used in patients with CA because of the invasiveness and the absence of high-quality data on its effect on outcome. We conducted a systematic review of experimental and clinical evidence to understand the role of PbtO2 in monitoring brain oxygenation in HIBI after CA and the effect of targeted PbtO2 therapy on outcomes. METHODS: The search was conducted using four search engines (PubMed, Scopus, Embase, and Cochrane), using the Boolean operator to combine mesh terms such as PbtO2, CA, and HIBI. RESULTS: Among 1,077 records, 22 studies were included (16 experimental studies and six clinical studies). In experimental studies, PbtO2 was mainly adopted to assess the impact of gas exchanges, drugs, or systemic maneuvers on brain oxygenation. In human studies, PbtO2 was rarely used to monitor the brain oxygen tension in patients with CA and HIBI. PbtO2 values had no clear association with patients' outcomes, but in the experimental studies, brain tissue hypoxia was associated with increased inflammation and neuronal damage. CONCLUSIONS: Further studies are needed to validate the effect and the threshold of PbtO2 associated with outcome in patients with CA, as well as to understand the physiological mechanisms influencing PbtO2 induced by gas exchanges, drug administration, and changes in body positioning after CA.

Urinary oxygen tension and its role in predicting acute kidney injury: A narrative review.

Acute kidney injury occurs frequently in the perioperative setting. The renal medulla often endures hypoxia or hypoperfusion and is susceptible to the imbalance between oxygen supply and demand due to the nature of renal blood flow distribution and metabolic rate in the kidney. The current available evidence demonstrated that the urine oxygen pressure is proportional to the variations of renal medullary tissue oxygen pressure. Thus, urine oxygenation can be a candidate for reflecting the change of oxygen in the renal medulla. In this review, we discuss the basic physiology of acute kidney injury, as well as techniques for monitoring urine oxygen tension, confounding factors affecting the reliable measurement of urine oxygen tension, and its clinical use, highlighting its potential role in early detection and prevention of acute kidney injury.

Impact of oxygen tension during in vitro maturation: a sibling-oocyte prospective double-blinded study.

OBJECTIVE: To determine whether oxygen (O2) tension (20% vs. 5%) has an impact on oocyte maturation rates and morphology during in vitro maturation (IVM). DESIGN: A prospective, observational, monocentric, sibling-oocyte study. SETTING: University Hospital. PATIENTS: A total of 143 patients who underwent IVM for fertility preservation purposes from November 2016 to April 2021 were analyzed. Patients were included when ≥2 cumulus-oocyte complexes (COCs) were retrieved. The cohort of COCs obtained for each patient was randomly split into two groups: group 20% O2 and group 5% O2. INTERVENTION: Cumulus-oocyte complexes were incubated for 48 hours either under 5% O2 or 20% O2. After 24 and 48 hours of culture, every oocyte was assessed for maturity and morphology, to estimate oocyte quality. Morphology was evaluated considering six parameters (shape, size, ooplasm, perivitelline space, zona pellucida, and polar body characteristics), giving a total oocyte score ranging from -6 to +6. MAIN OUTCOME MEASURES: Maturation rates and total oocyte scores were compared using paired-sample analysis between group 20% O2 and group 5% O2. RESULTS: Patient median age was 31.4 [28.1-35.2] years-old. The mean serum antimüllerian hormone levels and antral follicle count were 3.2 ± 2.3 ng/mL and 27.2 ± 16.0 follicles, respectively. A mean of 10.7 COCs per cycle were retrieved, leading to 6.1 ± 2.4 metaphase II oocytes vitrified (total maturation rate = 57.3%; 991 metaphase II oocytes/1,728 COCs). A total of 864 COCs were included in each group. Oocyte maturation rates were not different between the two groups (group 20% O2: 56.82% vs. group 5% O2: 57.87%, respectively). Regarding oocyte morphology, the mean total oocyte score was significantly higher in group 5% O2 compared with group 20% O2 (3.44 ± 1.26 vs. 3.16 ± 1.32, P=.014). CONCLUSION: As culture under low O2 tension (5% O2) improves oocyte morphology IVM, our results suggest that culture under hypoxia should be standardized. Additional studies are warranted to assess the impact of O2 tension on oocyte maturation and the benefit of IVM under low O2 tension for embryo culture after utilization of frozen material.

Temporal Statistical Relationship between Regional Cerebral Oxygen Saturation (rSO2) and Brain Tissue Oxygen Tension (PbtO2) in Moderate-to-Severe Traumatic Brain Injury: A Canadian High Resolution-TBI (CAHR-TBI) Cohort Study.

Brain tissue oxygen tension (PbtO2) has emerged as a cerebral monitoring modality following traumatic brain injury (TBI). Near-infrared spectroscopy (NIRS)-based regional cerebral oxygen saturation (rSO2) can non-invasively examine cerebral oxygen content and has the potential for high spatial resolution. Past studies examining the relationship between PbtO2 and NIRS-based parameters have had conflicting results with varying degrees of correlation. Understanding this relationship will help guide multimodal monitoring practices and impact patient care. The aim of this study is to examine the relationship between PbtO2 and rSO2 in a cohort of TBI patients by leveraging contemporary statistical methods. A multi-institutional retrospective cohort study of prospectively collected data was performed. Moderate-to-severe adult TBI patients were included with concurrent rSO2 and PbtO2 monitoring during their stay in the intensive care unit (ICU). The high-resolution data were analyzed utilizing time series techniques to examine signal stationarity as well as the cross-correlation relationship between the change in PbtO2 and the change in rSO2 signals. Finally, modeling of the change in PbtO2 by the change in rSO2 was attempted utilizing linear methods that account for the autocorrelative nature of the data signals. A total of 20 subjects were included in the study. Cross-correlative analysis found that changes in PbtO2 were most significantly correlated with changes in rSO2 one minute earlier. Through mixed-effects and time series modeling of parameters, changes in rSO2 were found to often have a statistically significant linear relationship with changes in PbtO2 that occurred a minute later. However, changes in rSO2 were inadequate to predict changes in PbtO2. In this study, changes in PbtO2 were found to correlate most with changes in rSO2 approximately one minute earlier. While changes in rSO2 were found to contain information about future changes in PbtO2, they were not found to adequately model them. This strengthens the body of literature indicating that NIRS-based rSO2 is not an adequate substitute for PbtO2 in the management of TBI.

Intra-tidal PaO2 oscillations associated with mechanical ventilation: a pilot study to identify discrete morphologies in a porcine model.

BACKGROUND: Within-breath oscillations in arterial oxygen tension (PaO2) can be detected using fast responding intra-arterial oxygen sensors in animal models. These PaO2 signals, which rise in inspiration and fall in expiration, may represent cyclical recruitment/derecruitment and, therefore, a potential clinical monitor to allow titration of ventilator settings in lung injury. However, in hypovolaemia models, these oscillations have the potential to become inverted, such that they decline, rather than rise, in inspiration. This inversion suggests multiple aetiologies may underlie these oscillations. A correct interpretation of the various PaO2 oscillation morphologies is essential to translate this signal into a monitoring tool for clinical practice. We present a pilot study to demonstrate the feasibility of a new analysis method to identify these morphologies. METHODS: Seven domestic pigs (average weight 31.1 kg) were studied under general anaesthesia with muscle relaxation and mechanical ventilation. Three underwent saline-lavage lung injury and four were uninjured. Variations in PEEP, tidal volume and presence/absence of lung injury were used to induce different morphologies of PaO2 oscillation. Functional principal component analysis and k-means clustering were employed to separate PaO2 oscillations into distinct morphologies, and the cardiorespiratory physiology associated with these PaO2 morphologies was compared. RESULTS: PaO2 oscillations from 73 ventilatory conditions were included. Five functional principal components were sufficient to explain ≥ 95% of the variance of the recorded PaO2 signals. From these, five unique morphologies of PaO2 oscillation were identified, ranging from those which increased in inspiration and decreased in expiration, through to those which decreased in inspiration and increased in expiration. This progression was associated with the estimates of the first functional principal component (P < 0.001, R2 = 0.88). Intermediate morphologies demonstrated waveforms with two peaks and troughs per breath. The progression towards inverted oscillations was associated with increased pulse pressure variation (P = 0.03). CONCLUSIONS: Functional principal component analysis and k-means clustering are appropriate to identify unique morphologies of PaO2 waveform associated with distinct cardiorespiratory physiology. We demonstrated novel intermediate morphologies of PaO2 waveform, which may represent a development of zone 2 physiologies within the lung. Future studies of PaO2 oscillations and modelling should aim to understand the aetiologies of these morphologies.

Culture conditions in the IVF laboratory: state of the ART and possible new directions.

In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Always enough but never too much: the how and why of downregulating tissue oxygenation.

Cardiovascular regulation of tissue oxygenation is generally viewed as an anti-drop process that prevents tissue oxygen concentration from falling below some minimum. I propose that cardiovascular regulation is predominately an anti-rise process designed to downregulate oxygen delivery. This maintains an evolutionarily conserved, reduced intracellular environment to prevent oxidation of redox-sensitive regulatory protein thiols. A number of points support this hypothesis. First, oxygen is the only nutrient with a positive, fourfold diffusion gradient from the environment to systemic tissues, minimizing the likelihood that oxygen delivery is limited. Second, hemoglobin (Hb) retains oxygen unless offloading is absolutely necessary. The allosteric properties of Hb keep oxygen tightly bound until absolutely needed, and the Bohr shift, which favors offloading, is only transient and lost when metabolism is restored. Third, a myoglobin-like Hb (xHb) would offload all of its oxygen and could easily have evolved, but it did not. Fourth, oxygen-sensitive vasoconstrictors and hyperoxic-rarefaction prevent acute and chronic over perfusion. Fifth, Fåhraeus and Fåhraeus-Lindqvist effects reduce capillary hematocrit to minimize microcirculatory oxygen content. Sixth, venous blood remains 75% saturated, wasting 75% of cardiac output were an oxygen reserve not needed. Finally, xHb-containing red blood cells could be considerably smaller and thereby decrease Fåhraeus and Fåhraeus-Lindqvist effects and cardiac load. In summary, the capacity of the cardiovascular system to deliver oxygen to the tissues generally exceeds demand, and although maintenance of an oxygen delivery reserve is important, it is more important to prevent excess oxygen delivery.

Measuring Mitochondrial Oxygen Tension during Red Blood Cell Transfusion in Chronic Anemia Patients: A Pilot Study.

In light of the associated risks, the question has been raised whether the decision to give a blood transfusion should solely be based on the hemoglobin level. As mitochondria are the final destination of oxygen transport, mitochondrial parameters are suggested to be of added value. The aims of this pilot study were to investigate the effect of a red blood cell transfusion on mitochondrial oxygenation as measured by the COMET device in chronic anemia patients and to explore the clinical usability of the COMET monitor in blood transfusion treatments, especially the feasibility of performing measurements in an outpatient setting. To correct the effect of volume load on mitochondrial oxygenation, a red blood cell transfusion and a saline infusion were given in random order. In total, 21 patients were included, and this resulted in 31 observations. If patients participated twice, the order of infusion was reversed. In both the measurements wherein a blood transfusion was given first and wherein 500 mL of 0.9% saline was given first, the median mitochondrial oxygen tension decreased after red blood cell transfusion. The results of this study have strengthened the need for further research into the effect of blood transfusion tissue oxygenation and the potential role of mitochondrial parameters herein.