Oxygen Equilibration Dynamics in Assisted Reproductive Technology Embryo Culture Media

Oxygen Equilibration Dynamics in Assisted Reproductive Technology Embryo Culture Media | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oxygen Equilibration Dynamics in Assisted Reproductive Technology Embryo Culture Media Sanjana Kulkarni, Bailey K Morris, Sacha A Krieg, Thomas O’Leary, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8143184/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Optimal oxygen tension is essential for successful in vitro embryo culture in assisted reproductive technology (ART). Reduced oxygen levels (3–8%) improve embryo development by minimizing oxidative stress, however, limited knowledge exists about transient oxygen fluctuations during handling outside hypoxic incubators. This study aimed to quantify the kinetics of oxygen equilibration in embryo culture media under conditions designed to mimic common ART laboratory practices. Methods Embryo culture media droplets were prepared in room air and overlayed with light or heavy mineral oil. Dishes were equilibrated in a hypoxia chamber (5% O₂, 5% CO₂, 37°C), then transferred to an atmospheric incubator (18–19% O₂, 5% CO₂) for equilibration, and then this was repeated once more. Oxygen saturation was measured every 30 seconds using a fiber optic microprobe (PreSens, GmbH). Each experiment was replicated three times, yielding six equilibration and six reoxygenation curves. Data were fit to single-phase exponential models to calculate half-lives and rate constants. Results Media equilibrated from atmospheric to hypoxic conditions within 12 hours. Light oil overlays demonstrated faster equilibration (half-life 71 min) compared to heavy oil (half-life 116 min, p < 0.0001). Upon reoxygenation, oxygen saturation rose rapidly, with light oil droplets reoxygenating with a doubling time of 50 min and heavy oil in 78 min (p < 0.0001). Conclusion In ART media, hypoxic oxygen equilibration is a gradual process while reoxygenation is rapid. Oil viscosity significantly influences oxygen equilibration dynamics, with light oil permitting faster equilibration and reoxygenation. These findings underscore the importance of minimizing atmospheric exposure during routine handling and highlight the role of overlay oil in reducing transient oxygen fluctuations. ART Media Preparation Reactive Oxygen Species Oxygen Saturation Equilibration Media Equilibration Figures Figure 1 Figure 2 Figure 3 Introduction The success of in vitro embryo culture in assisted reproductive technology (ART) is critically dependent on the microenvironmental conditions to which embryos are exposed. A key component of this environment is oxygen, which plays a pivotal role in supporting embryonic development. In vivo , prior to implantation, embryos develop within the oviduct and uterus where the oxygen levels are considerably lower than atmospheric levels of 20–21% O 2 [ 1 , 2 ]. The physiological oxygen concentration in the female reproductive tract between ranges from 2% to 8% O 2 [ 3 , 4 ]. ART laboratories have adopted reduced oxygen culture systems, most commonly around 5% O 2, to more closely mimic the environment of the female reproductive tract [ 5 , 6 ]. Excessive oxygen exposure during cell culture has been implicated in impaired embryonic development through oxidative mechanisms [ 7 ]. At high oxygen tension (approximately 21% O 2 ), reactive oxygen species (ROS) are generated, leading to lipid peroxidation, protein modification, and DNA damage that ultimately compromise embryo viability [ 8 , 9 ]. Early studies published between 1969 and 1971 demonstrated that culturing mouse embryos in low oxygen improved viability [ 10 , 11 ]. Studies evaluating other species such as human, bovine, mouse, goat and pig have consistently shown that culture under hypoxic conditions reduces ROS generation, preserves mitochondrial integrity, and improves blastocyst formation and embryo morphology [ 12 – 16 ]. More recent studies in rhesus macaques have similarly reported that low-oxygen conditions (3–5% O₂) support follicle growth, survival, and sustained steroidogenic function in vitro compared with atmospheric oxygen [ 17 ]. In human embryo culture, the use of low oxygen tension to improve ART outcomes is generally well accepted. Several randomized controlled trials and meta-analyses have demonstrated improved blastocyst development, implantation, and cumulative live birth rates in human embryos cultured under 5% oxygen compared with atmospheric oxygen [ 18 , 19 ]. As a result, hypoxic incubation has become more common in many ART laboratories [ 20 ]. Despite understanding the importance of hypoxic environments in embryo culture, embryo manipulation often requires exposure to atmospheric conditions. The dynamic nature of oxygen exposure during routine laboratory handling remains poorly understood. While incubators maintain stable hypoxic conditions, embryos are transiently exposed to atmospheric oxygen during key manipulations such as gamete handling, fertilization checks, embryo assessments, and media preparation. These brief exposures may introduce ROS and undermine the protective effects of a low-oxygen environment. A fundamental gap exists in understanding how rapidly oxygen levels change when culture media transitions between atmospheric and hypoxic environments. The rate of oxygen equilibrium could impact embryos or oocytes as rapid reoxygenation may lead to prolonged exposure to nonphysiological oxygen concentrations, potentially affecting development and implantation. The objective of this study is to quantify the time required for ART culture media to equilibrate from atmospheric (21% O 2 ) to hypoxic (5% O 2 ) conditions and vice versa. To our knowledge, this is the first study to systematically quantify the oxygen equilibration dynamics in ART culture media. By characterizing these equilibration times, we aim to provide practical data that may inform laboratory workflows and help minimize potentially harmful oxygen fluctuations during embryo culture. Methods/Material Preparation of culture droplets Dishes were prepared at atmospheric oxygen levels at approximately 60 meters above sea level and at room temperature in a Baker Sterigard class II A/B3 biological safety cabinet under aseptic conditions. Embryo culture medium (GlobalTotal) was dispensed at room temperature into 60 mm polystyrene dish (Falcon 351007) in 30 µl sized droplets, 4 microdrops in each dish to mimic standard clinical embryology conditions. After the microdrops were dispensed, they were overlaid with 8 mL of mineral oil. Dishes were made with two different types of oil, heavy and light oil (LifeGuard). The dishes were made one at a time prior to addition of the oil to ensure that there was minimal evaporation. Three independent measurements for each series were conducted. The oil and the media were dispensed at room temperature and were not equilibrated before dish preparation. Oxygen measurements Changes in oxygen tension were measured every 30 seconds with a Microx4 oxygen meter equipped with a needle-type fiber optic oxygen sensing microprobe (PreSens, GmbH). Each probe was carefully inserted into the center of a culture droplet under mineral oil, avoiding bubble formation or contact with dish plastic. The sensing probes were mounted in a manual micromanipulator to ensure consistent placement of sensor in media droplet (Fig. 1 A). Prior to each measurement series the sensor was allowed to equilibrate in each condition for at least five minutes, before transferring dishes to the respective O 2 conditions. Figure 1 A Diagram of Oxygen Sensing Apparatus. Oxygen concentration monitored using Microx 4 Oxygen Sensor equipped with needle-type probe (Pst-7-flat broken tip, PreSens GmbH, Germany). Probe consists of fiber-optic-line capped with oxygen-sensitive sensor foil that changes its reflective angle with O2 concentration. Fiberoptic line is threaded through a 18 gauge needle and 1 mL tuberculin syringe. A PreSens manual micromanipulator holds the probe in position with sensor foil immersed in media droplet. Sensor was calibrated to 15% w/v sodium sulfite (0% O2) and air saturated water (21% O2). Experiment Workflow and Incubation Conditions Dishes were first created at room temperature in atmospheric oxygen conditions on the bench top. Dishes were then transferred to a hypoxic glove box incubator (CoyLabs, Inc.) set to 5% O₂, 5% CO₂, and 37°C. The hypoxic glove box was equipped with a continuous oxygen monitoring system to ensure stable gas composition. The chamber’s oxygen sensor was calibrated to pure nitrogen and atmospheric air prior to each measurement series. The droplet oxygen concentration was measured until O₂ equilibrated after approximately 12 hours. Following this, plate and probe were then removed to a standard cell culture incubator (NuAire TS Auto Flow CO 2 water jacketed incubator) equilibrated with atmospheric O₂ with an actual reading of 18–19% O₂ and 5% CO₂ incubator. The single droplets were monitored until saturation was reached (approximately 12 hours). Plates and probes were then transferred back to the hypoxia glove box incubator set to 5% O₂ and 5% CO₂. The plates were reoxygenated for a final time for approximately 12 hours until equilibration. (Fig. 1 B). Replication The complete experiment was performed three independent times for each oil type, with new dishes, media, and oil preparations. This yielded six hypoxic equilibration curves (three from transitions 21% to 5% O₂, 5% CO₂; three from 5% CO₂ in humidified air) and six reoxygenation curves (5% O₂, 5% CO₂ to 21% O₂, 5% CO₂ in humidified air) (Fig. 1 B). Data Analysis Oxygen concentration (% O 2 ) over time was exported from PreSens software. Curves were fitted to a single-phase exponential decay or single-phase association model in GraphPad Prism Software. Data was reported as mean with standard deviation and 95% confidence intervals. Reoxygenation and hypoxic equilibration half times were calculated using least squares fit algorithm in GraphPad Prism. Equilibration curves were compared using extra sum of squares F test with a p-value of < 0.05 considered significant. Reoxygenation dynamics changed quickly for light and heavy oil within the first hour, therefore further discrete points were analyzed at 5-, 10- and 15-minutes intervals using one way ANOVA with corrections for repeated measurements with Dunnett's multiple comparisons test in GraphPad Prism. Results Hypoxic equilibration of media Media droplets immersed in culture oil were equilibrated to hypoxic conditions as described in Material and Methods and illustrated in Fig. 1 . When dishes prepared on the benchtop (atmospheric, ~ 21% O2, n = 3) were transferred to the hypoxia chamber, complete equilibration to 5% O₂ took approximately 12 hours with light oil overlay (Fig. 2 A). Equilibration followed a single-phase decay curve, with a plateau of 5.434% O2 and a half-life of 71.03 minutes (CI 95%; 70.23 to 71.84 mins; Table 1 ). Decay curves for independent replicate experiments using heavy oil immersion (n = 3) demonstrated slower trajectory when transferred from atmospheric oxygen to a hypoxia chamber, with a plateau of 5.440% O2 and a half-life of 116.3 minutes (CI 95%; 114.4 to 118.2, Fig. 2 B and Table 1 ). Comparison of the decay curves using the extra sum of squares F-test demonstrated a statistically significant difference (P < 0.0001) between the light oil (K = 0.009759; CI 95% 0.009648 to 0.00987) and heavy oil preparations (K = 0.005961; 95% CI 0.005865 to 0.006058; Table 1 and Fig. 2 C). Table 1 Hypoxia Trends Light Oil Heavy Oil Atmosphere > 5% O 2 Half Life [mins]; (95% CI) 71.03; (70.23 to 71.84) 116.3; (114.4 to 118.2) K-value; (95% CI) 0.0098; (0.0096 to 0.0098) 0.0060; (0.0059 to 0.0061) Atmospheric Incubator > 5% O 2 Half Life; (95% CI) 76.76; (76.49 to 77.03) 98.3; (97.09 to 99.59) K-value; (95% CI) 0.0090; (0.0089 to 0.0091) 0.0071 (0.0070 to 0.0071) Similarly, when equilibrated dishes overlain with light oil were transferred to the hypoxia chamber from a standard humidified 5% CO2 incubator set to atmospheric conditions (n = 3, ~ 18–19% O2), equilibration was also complete in approximately 12 hours, and followed a single-phase decay curve with a plateau of 5.141% O2 and a half-life of 76.76 minutes (CI 95%; 76.49 to 77.03 mins; Fig. 2 D and Table 1 ). When droplets equilibrated in heavy oil were moved from the incubator to the hypoxia chamber (n = 3), oxygen measurements also followed single-phase decay curve with a plateau of 5.311% O2 and a half-life of 98.33 minutes (CI 95%; 97.09 to 99.59, Fig. 2 E and Table 1 ). The equilibration curves for these two series were significantly different (P < 0.0001, Fig. 2 F and Table 1 ). The light oil curve had a K-value of 0.009030 (CI 95%; 0.0089 to 0.0091) while heavy oil had a K-value of 0.007049 (CI 95%; 0.006960 to 0.007139). Reoxygenation Dynamics In both light oil and heavy oil media preparations, media was allowed to reach complete reoxygenation for approximately 12 hours. The doubling time for reoxygenation in the light oil was 50.22 minutes (CI 95%; 49.63 to 50.82 minutes) ( Table 2 ). Reoxygenation dynamics of light oil (n = 6) demonstrated a single-phase association curve with K-value of 0.01380 (CI 95%; 0.01364 to 0.01296; Fig. 3 A and Table 2 ). Comparatively, the reoxygenation doubling time for heavy oil was 78.49 minutes (CI 95%; 77.07 to 79.95, Table 2 ). The reoxygenation of the droplets immersed in heavy oil also demonstrated a single-phase association curve with a K value of 0.008831 (CI 95%; 0.008670 to 0.008993; Table 2 and Fig. 3 B). Comparison of reoxygenation curves with the extra sum of squares F test demonstrated significantly slower reoxygenation dynamics for the heavy oil dishes (P < 0.0001; Fig. 3 C and Table 2 ). Reoxygenation of media overlain by light oil in the humified incubator (~ 18–19% O2) was statistically significant immediately following transfer (t = 0 minutes), with a mean difference of − 0.8330 compared to hypoxic equilibration (CI 95%; -1.487 to − 0.1794) and a p-value of 0.0191 (Fig. 3 D and Table 3 ). The reoxygenation of media under heavy oil was at a slower rate than the light oil with statistically significant reoxygenation occurring 15 minutes after transfer from hypoxia, with a mean difference of − 1.590 (CI 95%; − 2.944 to − 0.2362) and a p-value 0.026 (Fig. 3 D and Table 4 ). Table 2 Reoxygenation Trends Light Oil Heavy Oil 5% O 2 > Atmospheric Incubator Doubling time [mins]; (95% CI) 50.22; (49.63 to 50.82) 78.49; (77.07 to 79.95) K-value; (95% CI) 0.0138; (0.0136 to 0.0129) 0.0088; (0.0087 to 0.0090) Table 3 Light Oil Reoxygenation Discrete points Dunnett's multiple comparisons test Mean diff. 95.00% CI of diff. Below threshold Adjusted P -Value Hypoxia vs. 0 -0.833 -1.487 to -0.179 Yes 0.0191 Hypoxia vs. 5 -1.385 -2.169 to -0.600 Yes 0.0046 Hypoxia vs. 10 -2.301 -3.279 to -1.324 Yes 0.0012 Hypoxia vs. 15 -3.197 -4.335 to -2.059 Yes 0.0005 Hypoxia vs. 20 -4.009 -5.258 to -2.761 Yes 0.0003 Hypoxia vs. 30 -5.392 -6.751 to -4.033 Yes <0.0001 Hypoxia vs. 45 -7.004 -8.245 to -5.764 Yes <0.0001 Hypoxia vs. 60 -8.263 -9.296 to -7.229 Yes <0.0001 Table 4 Heavy Oil Reoxygenation Discrete points Dunnett's multiple comparisons test Mean diff. 95.00% CI of diff. Below threshold Adjusted P-Value Hypoxia vs. 0 -0.563 -1.973 to 0.846 No 0.5646 Hypoxia vs. 5 -0.843 -2.678 to 0.994 No 0.4514 Hypoxia vs. 10 -1.149 -2.604 to 0.306 No 0.1140 Hypoxia vs. 15 -1.590 -2.944 to -0.236 Yes 0.0268 Hypoxia vs. 20 -2.163 -3.770 to -0.555 Yes 0.0153 Hypoxia vs. 30 -3.157 -5.215 to -1.099 Yes 0.0087 Hypoxia vs. 45 -4.459 -6.662 to -2.256 Yes 0.0025 Hypoxia vs. 60 -5.638 -7.860 to -3.417 Yes 0.0009 Discussion To the best of our knowledge, our study provides the first known, direct quantitation of oxygen equilibration and reoxygenation dynamics in embryo culture media using conditions designed to mimic routine ART laboratory practices. We demonstrated that oxygen exchange is gradual but predictable with equilibration under hypoxia. Our results also demonstrate that reoxygenation can occur rapidly with significant rises in O₂ within minutes of exposure to ambient air. Additionally, the choice of oil overlay significantly alters these dynamics, with light oil allowing faster equilibration and reoxygenation compared to heavy oil. The influence of oil type impacts buffering capacity and reoxygenation rates, creating a dilemma in oil choice for embryo culture. The intent of our experimental design was to closely reflect practices in embryology laboratories. During routine in vitro fertilization (IVF) procedures, media preparation is frequently performed on the benchtop in atmospheric oxygen, after which plates are transferred into low oxygen incubators to more closely mimic the physiologic environment in utero . The first portion of our experiment closely emulated this, as the dishes were prepared on the bench top then moved to a hypoxic incubator (Fig. 1 B). Similarly, oocytes and embryos are often removed from the hypoxic incubator for micromanipulation steps such as intracytoplasmic sperm injection (ICSI) or embryo biopsy, briefly exposing them to atmospheric oxygen before being returned to reduced oxygen [ 21 ]. Since embryo manipulation often takes place in atmospheric oxygen, to recreate the laboratory environment, the O2 sensor was equilibrated in the destination incubator, dishes were then transferred to a conventional 5% CO2, 37°C incubator and allowed to equilibrate overnight to atmospheric conditions to measure reoxygenation dynamics of embryo culture media (Fig. 1 B). Some ART labs perform their dish preparation in a preequilibrated incubator (18–19% O 2 ) then moved into a hypoxic incubator. Therefore, we assessed hypoxia equilibrium when embryo culture media was moved from an atmospheric incubator to a hypoxia chamber (5% O 2 ). In our experiment the dishes were then returned to the hypoxia chamber for at least 12-hours and then reoxygenated to mimic the routine handling steps, allowing us to quantify the equilibration dynamics of culture media under realistic laboratory conditions. In our study we used a hypoxic glove box incubator (hypoxia chamber) maintained at 5% O 2 which prevented reoxygenation artifacts during the process of manipulating the oxygen probe. This chamber also maintained constant temperature, which can also influence oxygen sensor readings [ 4 ]. A primary goal of our study was to mimic culture conditions without inserting the additional influence of embryo metabolism. Our work highlights how fluctuations in environmental oxygen saturation could influence the stability of the microenvironment surrounding embryos. By characterizing the kinetics of oxygen equilibration across different manipulations, our study underscores the potential for transient reoxygenation events to affect embryo culture outcomes. This provides valuable insight into best practices for minimizing oxygen fluctuations, reinforcing the importance of consistent hypoxic culture conditions in optimizing IVF success rates. Both the light and heavy oil equilibrations followed single-phase decay curves, but the half-life to equilibration in light oil was significantly lower than the half-life for the heavy oil. These results indicate that longer equilibration times should be considered in standard laboratory workflows that use heavy oil. This highlights the impact of oil viscosity and density on oxygen diffusion, which had been suggested in theory but here is directly measured [ 22 ]. The result of our study has relevant implications for ART practice. Notably, oxygen concentrations used for embryo culture vary considerably across clinics and regions. Many laboratories continue to culture embryos at atmospheric oxygen (20%) despite recommendations supporting reduced oxygen conditions (5%) to more closely reflect the physiologic reproductive environment [ 23 , 24 ]. Culturing embryos at elevated oxygen has been shown to cause massive gene deregulation in mice, including genes required for cell growth and gastrulation [ 25 ]. Similarly, bovine embryos cultured under elevated oxygen exhibit increased DNA methylation and reduced blastocyst formation rates [ 25 , 26 ]. Exposure to atmospheric oxygen further enhances the activity of oxygen-dependent enzymes, leading to accelerated ROS generation, heightened oxidative stress, and prolonged embryo development [ 27 , 28 ]. This dynamic is particularly concerning given the known sensitivity of preimplantation embryos to oxygen fluctuations and the potential for reactive oxygen species (ROS) mediated damage [ 29 ]. ROS are often generated in vitro and rise if oxygen tensions of the media increases, impairing embryo competence and viability [ 30 ] [ 31 ]. This can impair embryo competence and viability [ 30 ]. Our study has some limitations. First, our kinetic data was derived from a media only system without the influence of embryo metabolism. Embryos may alter oxygen dynamics due to metabolic waste production and cellular oxygen consumption which may accelerate or delay equilibration rates [ 29 ]. Investigating oxygen kinetics in the presence of embryos, as well as directly measuring ROS levels, could provide more biologically relevant insights. Furthermore, only one formulation of culture media was tested therefore this data may not be generalizable for all other media types. Further studies would be recommended to compare multiple media types. Additionally, media droplets were pipetted into the dish to mimic standard embryo culture conditions, but only one of the droplets could be measured at one time due the oxygen monitor only having one sensor input. Attempts to measure changes in all four droplets in sequence resulted in equilibration artifacts due to the transition of the sensor between droplets. More consistent readings were achieved by fixing the probe in one droplet with a micromanipulator (Fig. 1 A). Incubator space also did not allow for more than one micromanipulator per experiment. Finally, our study did not evaluate the impact of the shift in oxygenation on the embryo itself. It is not yet known if short term shifts in oxygen have an impact on embryo quality. Taken together, these results argue strongly for minimizing atmospheric exposure during all phases of embryo culture and for implementing hypoxic workstations or isolettes wherever feasible. Moreover, our direct measurements of equilibration kinetics offer embryologists practical guidance on how quickly reoxygenation occurs and how oil overlay choices modulate this process. By defining these parameters, our study provides practical and quantitative benchmarks for reoxygenation kinetics, that contribute to the development of evidence-based best practices aimed at reducing oxygen-related artifacts, safeguarding embryo development, and ultimately improving IVF outcomes. Declarations Author Contribution All authors contributed to the study conception and design. The experiment was conducted by Adam Krieg and Thomas O’Leary. Data analysis was performed by Adam Krieg and Sanjana Kulkarni. The first draft of the manuscript was written by Sanjana Kulkarni and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement We acknowledge support from the National Institutes of Health R21HD094983 (AJK) and R01HD082208 (AJK) for this manuscript. Data Availability All data supporting the findings of this study are available within the paper. References Fischer, B. and B.D. Bavister, Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil, 1993. 99 (2): p. 673-9. Maas, D.H., B.T. Storey, and L. Mastroianni, Jr., Oxygen tension in the oviduct of the rhesus monkey (Macaca mulatta). Fertil Steril, 1976. 27 (11): p. 1312-7. Sciorio, R. and G.D. Smith, Embryo culture at a reduced oxygen concentration of 5%: a mini review. Zygote, 2019. 27 (6): p. 355-361. 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Li, W., et al., High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression. Reprod Fertil Dev, 2016. 28 (7): p. 948-959. Guerin, P., S. El Mouatassim, and Y. Menezo, Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update, 2001. 7 (2): p. 175-89. Kovacic, B. and V. Vlaisavljevic, Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes. Reprod Biomed Online, 2008. 17 (2): p. 229-36. Cooke, M.S., et al., Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J, 2003. 17 (10): p. 1195-214. Cobley, J.N., Mechanisms of Mitochondrial ROS Production in Assisted Reproduction: The Known, the Unknown, and the Intriguing. Antioxidants (Basel), 2020. 9 (10). Shields, H.J., A. Traa, and J.M. Van Raamsdonk, Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies. Front Cell Dev Biol, 2021. 9 : p. 628157. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 14 Dec, 2025 Reviewers invited by journal 10 Dec, 2025 Editor assigned by journal 22 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 18 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8143184","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560380164,"identity":"01d1fd76-fa75-4253-ac48-bf723705c2e2","order_by":0,"name":"Sanjana 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Krieg","email":"","orcid":"","institution":"Oregon Health and Science University","correspondingAuthor":false,"prefix":"","firstName":"Sacha","middleName":"A","lastName":"Krieg","suffix":""},{"id":560380167,"identity":"b031f1de-9a81-43dd-9ae2-edab8bf2f6d4","order_by":3,"name":"Thomas O’Leary","email":"","orcid":"","institution":"Oregon Health and Science University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"O’Leary","suffix":""},{"id":560380168,"identity":"12459101-c4f7-43c6-8a38-3762a4bce2e2","order_by":4,"name":"Adam Krieg","email":"","orcid":"","institution":"Oregon Health and Science University","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Krieg","suffix":""}],"badges":[],"createdAt":"2025-11-18 08:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8143184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8143184/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98437423,"identity":"f5e8f4d5-2ca5-4045-8769-b557ae4ae5ea","added_by":"auto","created_at":"2025-12-17 16:57:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":902818,"visible":true,"origin":"","legend":"","description":"","filename":"FinalManuscript11.17.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/215e4adb8ca2e6e3ba5c9b0d.docx"},{"id":98437289,"identity":"db0f21a1-d047-4cdf-aebd-5f30bc525014","added_by":"auto","created_at":"2025-12-17 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13:37:54","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81235,"visible":true,"origin":"","legend":"","description":"","filename":"305a3c7169a347f4b9075b08604b981a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/739ca73d755caaca057270c6.xml"},{"id":98317240,"identity":"6a5a1d82-7f1f-4498-8414-58b167586eec","added_by":"auto","created_at":"2025-12-16 13:37:55","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88903,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/93fab62969d59f3fa735d760.html"},{"id":98317225,"identity":"6334ad72-205b-41b0-aef0-562925e59579","added_by":"auto","created_at":"2025-12-16 13:37:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Diagram of Oxygen Sensing Apparatus. Oxygen concentration monitored using Microx 4 Oxygen Sensor equipped with needle-type probe (Pst-7-flat broken tip, PreSens GmbH, Germany). Probe consists of fiber-optic-line capped with oxygen-sensitive sensor foil that changes its reflective angle with O2 concentration. Fiberoptic line is threaded through a 18 gauge needle and 1 mL tuberculin syringe. A PreSens manual micromanipulator holds the probe in position with sensor foil immersed in media droplet. Sensor was calibrated to 15% w/v sodium sulfite (0% O2) and air saturated water (21% O2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e: Diagram of Experimental Timelines. Plates containing oil-embedded media droplets were transferred to hypoxia glovebox incubators (5% O2, 5% CO2, ~90% N2, 37°C, CoyLabs) for 12-18 hours and measured using the Microx 4 as in Figure 1. After transfer and equilibration of the O2 sensor, dish was transferred to a conventional 5% CO2, 37°C incubator and allowed to reoxygenate overnight (12-18 hours). Dish was returned to the Hypoxia Chamber for 16-24 hours and then reoxygenated. O2 measurements were collected every 30 seconds throughout the duration of the experiments.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/d11b7669918e9cc9910d2100.png"},{"id":98317224,"identity":"5194985c-0a41-4e62-877b-747104e0d01e","added_by":"auto","created_at":"2025-12-16 13:37:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of Atmospheric O2 and Culture Oil Density on the Dynamics of Hypoxic Equilibrium. A.\u003c/strong\u003e \u003cstrong\u003eHypoxic Equilibration in Light Oil from Atmospheric Conditions. \u003c/strong\u003eOxygen concentration was measured in embryo culture media overlayed with light oil as described in Materials and Methods. The blue line represents the mean oxygen concentration over time after the media was transferred from the benchtop (~20% O₂) into the hypoxia chamber (~ 5% O₂, N=3 independent replicates, SE shown in light blue). \u003cstrong\u003eB. Hypoxic Equilibration in Heavy Oil from Atmospheric Conditions. \u003c/strong\u003eOxygen concentration was measured in embryo culture media overlayed with heavy oil as described in Panel A. The black line represents the mean oxygen concentration over time following transfer to the hypoxia chamber. (N=3, independent replicates, SE shown in gray). \u0026nbsp;\u003cstrong\u003eC.\u003c/strong\u003e \u003cstrong\u003eComparison of Atmospheric-to-Hypoxia Equilibration Curves. \u003c/strong\u003eData from Panels A (blue) and B (black) were fit to single-phase decay curves and compared using extra sum of squares F-test as described in Materials and Methods (p\u0026lt;0.001). \u003cstrong\u003eD. Hypoxic Equilibration in Light Oil from Standard Incubator Conditions. \u003c/strong\u003eOxygen concentration was measured in embryo culture media overlayed with light oil. The blue line represents the mean oxygen concentration over time after the media was transferred from a standard incubator (~18-19% O₂) to a hypoxia chamber (N=3 independent replicates, SE shown in light blue). \u003cstrong\u003eE. Hypoxic Equilibration in Heavy Oil from Standard Incubator. \u0026nbsp;\u003c/strong\u003eThe black line represents the mean oxygen concentration over time after media immersed in heavy oil was transferred from a standard incubator to a hypoxia chamber (N=3 independent replicates, SE shown in gray). \u003cstrong\u003eF. Comparison of Incubator-to-Hypoxia Equilibration Curves. \u003c/strong\u003eData from Panels D (blue) and E (black) were fit to single-phase decay curves and compared using extra sum of squares F-test as described in Materials and Methods (p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/6b09c947e3310c4be38bc587.png"},{"id":98317227,"identity":"505ab5d5-428a-458a-9feb-3f3ddcd1b3ad","added_by":"auto","created_at":"2025-12-16 13:37:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of Oil Density on Dynamics of Reoxygenation. A.\u003c/strong\u003e \u003cstrong\u003eReoxygenation Dynamics in Light Oil. \u003c/strong\u003eOxygen concentration was measured in embryo culture media overlayed with light oil as described in Materials and Methods. The red line represents the mean oxygen concentration over time after hypoxic media (~5% O₂) transferred into an atmospheric incubator (~ 18-19% O₂; N=6 independent replicates, SE shown in light red). \u003cstrong\u003eB. Reoxygenation Dynamics in Heavy Oil. \u003c/strong\u003eOxygen concentration was measured in embryo culture media overlayed with heavy oil. The black line represents the mean oxygen concentration over time after the media was transferred into an atmospheric incubator (~ 18-19% O₂; N=6 independent replicates, SE shown in gray). \u003cstrong\u003eC. Comparison of Reoxygenation Curves. \u003c/strong\u003eData from Panel A (light oil, blue) and Panel B (heavy oil, black) was fit to single-phase decay curves as described in Materials and Methods and compared using extra sum of squares F-test as described in Materials and Methods (p\u0026lt;0.001). \u003cstrong\u003eD. Reoxygenation at Discrete Time Points in Light Oil.\u0026nbsp; \u003c/strong\u003eAverage O\u003csub\u003e2\u003c/sub\u003e% measurements were plotted at discrete timepoints for up to 60 minutes following reoxygenation of embryo culture overlain in light oil. Data represents the mean concentration ± 95% confidence intervals compared to Hypoxia using One-Way ANOVA of repeated measurements with Dunnet’s multiple comparison test (* = P \u0026lt; 0.05, ** = P \u0026lt; 0.01, *** = P \u0026lt; 0.001, **** = P \u0026lt; 0.0001). \u003cstrong\u003eE. Reoxygenation at Discrete Points in Heavy Oil. \u003c/strong\u003eData represents the mean concentration ± 95% confidence intervals compared to Hypoxia using One-Way ANOVA of repeated measurements with Dunnet’s multiple comparison test (* = P \u0026lt; 0.05, ** = P \u0026lt; 0.01, *** = P \u0026lt; 0.001).\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/493dc48a9cff6b01261bb544.png"},{"id":98774568,"identity":"35562b32-d504-4548-8f95-0bc460543f87","added_by":"auto","created_at":"2025-12-22 12:01:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1412222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8143184/v1/ce576ec2-85f0-4c14-917b-b482fba7d5fb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oxygen Equilibration Dynamics in Assisted Reproductive Technology Embryo Culture Media","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe success of \u003cem\u003ein vitro\u003c/em\u003e embryo culture in assisted reproductive technology (ART) is critically dependent on the microenvironmental conditions to which embryos are exposed. A key component of this environment is oxygen, which plays a pivotal role in supporting embryonic development. \u003cem\u003eIn vivo\u003c/em\u003e, prior to implantation, embryos develop within the oviduct and uterus where the oxygen levels are considerably lower than atmospheric levels of 20\u0026ndash;21% O\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The physiological oxygen concentration in the female reproductive tract between ranges from 2% to 8% O\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ART laboratories have adopted reduced oxygen culture systems, most commonly around 5% O\u003csub\u003e2,\u003c/sub\u003e to more closely mimic the environment of the female reproductive tract [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExcessive oxygen exposure during cell culture has been implicated in impaired embryonic development through oxidative mechanisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At high oxygen tension (approximately 21% O\u003csub\u003e2\u003c/sub\u003e), reactive oxygen species (ROS) are generated, leading to lipid peroxidation, protein modification, and DNA damage that ultimately compromise embryo viability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Early studies published between 1969 and 1971 demonstrated that culturing mouse embryos in low oxygen improved viability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies evaluating other species such as human, bovine, mouse, goat and pig have consistently shown that culture under hypoxic conditions reduces ROS generation, preserves mitochondrial integrity, and improves blastocyst formation and embryo morphology [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. More recent studies in rhesus macaques have similarly reported that low-oxygen conditions (3\u0026ndash;5% O₂) support follicle growth, survival, and sustained steroidogenic function \u003cem\u003ein vitro\u003c/em\u003e compared with atmospheric oxygen [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn human embryo culture, the use of low oxygen tension to improve ART outcomes is generally well accepted. Several randomized controlled trials and meta-analyses have demonstrated improved blastocyst development, implantation, and cumulative live birth rates in human embryos cultured under 5% oxygen compared with atmospheric oxygen [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As a result, hypoxic incubation has become more common in many ART laboratories [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite understanding the importance of hypoxic environments in embryo culture, embryo manipulation often requires exposure to atmospheric conditions. The dynamic nature of oxygen exposure during routine laboratory handling remains poorly understood. While incubators maintain stable hypoxic conditions, embryos are transiently exposed to atmospheric oxygen during key manipulations such as gamete handling, fertilization checks, embryo assessments, and media preparation. These brief exposures may introduce ROS and undermine the protective effects of a low-oxygen environment. A fundamental gap exists in understanding how rapidly oxygen levels change when culture media transitions between atmospheric and hypoxic environments. The rate of oxygen equilibrium could impact embryos or oocytes as rapid reoxygenation may lead to prolonged exposure to nonphysiological oxygen concentrations, potentially affecting development and implantation.\u003c/p\u003e \u003cp\u003eThe objective of this study is to quantify the time required for ART culture media to equilibrate from atmospheric (21% O\u003csub\u003e2\u003c/sub\u003e) to hypoxic (5% O\u003csub\u003e2\u003c/sub\u003e) conditions and vice versa. To our knowledge, this is the first study to systematically quantify the oxygen equilibration dynamics in ART culture media. By characterizing these equilibration times, we aim to provide practical data that may inform laboratory workflows and help minimize potentially harmful oxygen fluctuations during embryo culture.\u003c/p\u003e"},{"header":"Methods/Material","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of culture droplets\u003c/h2\u003e \u003cp\u003eDishes were prepared at atmospheric oxygen levels at approximately 60 meters above sea level and at room temperature in a Baker Sterigard class II A/B3 biological safety cabinet under aseptic conditions. Embryo culture medium (GlobalTotal) was dispensed at room temperature into 60 mm polystyrene dish (Falcon 351007) in 30 \u0026micro;l sized droplets, 4 microdrops in each dish to mimic standard clinical embryology conditions. After the microdrops were dispensed, they were overlaid with 8 mL of mineral oil. Dishes were made with two different types of oil, heavy and light oil (LifeGuard). The dishes were made one at a time prior to addition of the oil to ensure that there was minimal evaporation. Three independent measurements for each series were conducted. The oil and the media were dispensed at room temperature and were not equilibrated before dish preparation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOxygen measurements\u003c/h3\u003e\n\u003cp\u003e Changes in oxygen tension were measured every 30 seconds with a Microx4 oxygen meter equipped with a needle-type fiber optic oxygen sensing microprobe (PreSens, GmbH). Each probe was carefully inserted into the center of a culture droplet under mineral oil, avoiding bubble formation or contact with dish plastic. The sensing probes were mounted in a manual micromanipulator to ensure consistent placement of sensor in media droplet (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Prior to each measurement series the sensor was allowed to equilibrate in each condition for at least five minutes, before transferring dishes to the respective O\u003csub\u003e2\u003c/sub\u003e conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003c/strong\u003e \u003cp\u003eDiagram of Oxygen Sensing Apparatus. Oxygen concentration monitored using Microx 4 Oxygen Sensor equipped with needle-type probe (Pst-7-flat broken tip, PreSens GmbH, Germany). Probe consists of fiber-optic-line capped with oxygen-sensitive sensor foil that changes its reflective angle with O2 concentration. Fiberoptic line is threaded through a 18 gauge needle and 1 mL tuberculin syringe. A PreSens manual micromanipulator holds the probe in position with sensor foil immersed in media droplet. Sensor was calibrated to 15% w/v sodium sulfite (0% O2) and air saturated water (21% O2).\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eExperiment Workflow and Incubation Conditions\u003c/h3\u003e\n\u003cp\u003eDishes were first created at room temperature in atmospheric oxygen conditions on the bench top. Dishes were then transferred to a hypoxic glove box incubator (CoyLabs, Inc.) set to 5% O₂, 5% CO₂, and 37\u0026deg;C. The hypoxic glove box was equipped with a continuous oxygen monitoring system to ensure stable gas composition. The chamber\u0026rsquo;s oxygen sensor was calibrated to pure nitrogen and atmospheric air prior to each measurement series. The droplet oxygen concentration was measured until O₂ equilibrated after approximately 12 hours. Following this, plate and probe were then removed to a standard cell culture incubator (NuAire TS Auto Flow CO\u003csub\u003e2\u003c/sub\u003e water jacketed incubator) equilibrated with atmospheric O₂ with an actual reading of 18\u0026ndash;19% O₂ and 5% CO₂ incubator. The single droplets were monitored until saturation was reached (approximately 12 hours). Plates and probes were then transferred back to the hypoxia glove box incubator set to 5% O₂ and 5% CO₂. The plates were reoxygenated for a final time for approximately 12 hours until equilibration. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003eReplication\u003c/h3\u003e\n\u003cp\u003eThe complete experiment was performed three independent times for each oil type, with new dishes, media, and oil preparations. This yielded six hypoxic equilibration curves (three from transitions 21% to 5% O₂, 5% CO₂; three from 5% CO₂ in humidified air) and six reoxygenation curves (5% O₂, 5% CO₂ to 21% O₂, 5% CO₂ in humidified air) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eOxygen concentration (% O\u003csub\u003e2\u003c/sub\u003e) over time was exported from PreSens software. Curves were fitted to a single-phase exponential decay or single-phase association model in GraphPad Prism Software. Data was reported as mean with standard deviation and 95% confidence intervals. Reoxygenation and hypoxic equilibration half times were calculated using least squares fit algorithm in GraphPad Prism. Equilibration curves were compared using extra sum of squares F test with a p-value of \u0026lt;\u0026thinsp;0.05 considered significant. Reoxygenation dynamics changed quickly for light and heavy oil within the first hour, therefore further discrete points were analyzed at 5-, 10- and 15-minutes intervals using one way ANOVA with corrections for repeated measurements with Dunnett's multiple comparisons test in GraphPad Prism.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch3\u003eHypoxic equilibration of media\u003c/h3\u003e\n\u003cp\u003eMedia droplets immersed in culture oil were equilibrated to hypoxic conditions as described in Material and Methods and illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. When dishes prepared on the benchtop (atmospheric, ~\u0026thinsp;21% O2, n\u0026thinsp;=\u0026thinsp;3) were transferred to the hypoxia chamber, complete equilibration to 5% O₂ took approximately 12 hours with light oil overlay (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Equilibration followed a single-phase decay curve, with a plateau of 5.434% O2 and a half-life of 71.03 minutes (CI 95%; 70.23 to 71.84 mins; \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e). Decay curves for independent replicate experiments using heavy oil immersion (n\u0026thinsp;=\u0026thinsp;3) demonstrated slower trajectory when transferred from atmospheric oxygen to a hypoxia chamber, with a plateau of 5.440% O2 and a half-life of 116.3 minutes (CI 95%; 114.4 to 118.2, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e). Comparison of the decay curves using the extra sum of squares F-test demonstrated a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) between the light oil (K\u0026thinsp;=\u0026thinsp;0.009759; CI 95% 0.009648 to 0.00987) and heavy oil preparations (K\u0026thinsp;=\u0026thinsp;0.005961; 95% CI 0.005865 to 0.006058; \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 1 Hypoxia Trends\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLight Oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeavy Oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAtmosphere\u0026thinsp;\u0026gt;\u0026thinsp;5% O\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHalf Life [mins]; (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.03; (70.23 to 71.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.3; (114.4 to 118.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK-value; (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0098; (0.0096 to 0.0098)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0060; (0.0059 to 0.0061)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAtmospheric Incubator\u0026thinsp;\u0026gt;\u0026thinsp;5% O\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHalf Life; (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.76; (76.49 to 77.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.3; (97.09 to 99.59)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK-value; (95% CI)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0090; (0.0089 to 0.0091)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0071 (0.0070 to 0.0071)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSimilarly, when equilibrated dishes overlain with light oil were transferred to the hypoxia chamber from a standard humidified 5% CO2 incubator set to atmospheric conditions (n\u0026thinsp;=\u0026thinsp;3, ~\u0026thinsp;18\u0026ndash;19% O2), equilibration was also complete in approximately 12 hours, and followed a single-phase decay curve with a plateau of 5.141% O2 and a half-life of 76.76 minutes (CI 95%; 76.49 to 77.03 mins; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e). When droplets equilibrated in heavy oil were moved from the incubator to the hypoxia chamber (n\u0026thinsp;=\u0026thinsp;3), oxygen measurements also followed single-phase decay curve with a plateau of 5.311% O2 and a half-life of 98.33 minutes (CI 95%; 97.09 to 99.59, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cstrong\u003eTable\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e). The equilibration curves for these two series were significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e). The light oil curve had a K-value of 0.009030 (CI 95%; 0.0089 to 0.0091) while heavy oil had a K-value of 0.007049 (CI 95%; 0.006960 to 0.007139).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eReoxygenation Dynamics\u003c/h2\u003e\n \u003cp\u003eIn both light oil and heavy oil media preparations, media was allowed to reach complete reoxygenation for approximately 12 hours. The doubling time for reoxygenation in the light oil was 50.22 minutes (CI 95%; 49.63 to 50.82 minutes) (\u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e). Reoxygenation dynamics of light oil (n\u0026thinsp;=\u0026thinsp;6) demonstrated a single-phase association curve with K-value of 0.01380 (CI 95%; 0.01364 to 0.01296; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e). Comparatively, the reoxygenation doubling time for heavy oil was 78.49 minutes (CI 95%; 77.07 to 79.95, \u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e). The reoxygenation of the droplets immersed in heavy oil also demonstrated a single-phase association curve with a K value of 0.008831 (CI 95%; 0.008670 to 0.008993; \u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Comparison of reoxygenation curves with the extra sum of squares F test demonstrated significantly slower reoxygenation dynamics for the heavy oil dishes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eReoxygenation of media overlain by light oil in the humified incubator (~\u0026thinsp;18\u0026ndash;19% O2) was statistically significant immediately following transfer (t\u0026thinsp;=\u0026thinsp;0 minutes), with a mean difference of \u0026minus;\u0026thinsp;0.8330 compared to hypoxic equilibration (CI 95%; -1.487 to \u0026minus;\u0026thinsp;0.1794) and a p-value of 0.0191 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD and Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The reoxygenation of media under heavy oil was at a slower rate than the light oil with statistically significant reoxygenation occurring 15 minutes after transfer from hypoxia, with a mean difference of \u0026minus;\u0026thinsp;1.590 (CI 95%; \u0026minus;\u0026thinsp;2.944 to \u0026minus;\u0026thinsp;0.2362) and a p-value 0.026 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD and Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eReoxygenation Trends\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"450\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLight Oil\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeavy Oil\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5% O\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e\u0026gt; Atmospheric Incubator\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eDoubling time [mins]; (95% CI)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e50.22; (49.63 to 50.82)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e78.49; (77.07 to 79.95)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eK-value; (95% CI)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.0138; (0.0136 to 0.0129)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.0088; (0.0087 to 0.0090)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"593\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"bottom\" style=\"width: 593px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eLight Oil Reoxygenation Discrete points\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDunnett\u0026apos;s multiple comparisons test\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean diff.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95.00% CI of diff.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBelow threshold\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdjusted P -Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-0.833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-1.487 to -0.179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.0191\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-1.385\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-2.169 to -0.600\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.0046\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-2.301\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-3.279 to -1.324\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.0012\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-3.197\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-4.335 to -2.059\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.0005\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-4.009\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-5.258 to -2.761\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.0003\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-5.392\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-6.751 to -4.033\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-7.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-8.245 to -5.764\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHypoxia vs. 60\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-8.263\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-9.296 to -7.229\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eHeavy Oil Reoxygenation Discrete points\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDunnett\u0026apos;s multiple comparisons test\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean diff.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95.00% CI of diff.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBelow threshold\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdjusted P-Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 0 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-1.973 to 0.846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eNo\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.5646\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 5 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-2.678 to 0.994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eNo\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.4514\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-1.149\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-2.604 to 0.306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eNo\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.1140\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-1.590\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-2.944 to -0.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.0268\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-2.163\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-3.770 to -0.555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.0153\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-3.157\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-5.215 to -1.099\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.0087\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-4.459\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-6.662 to -2.256\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.0025\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 192px;\"\u003e\n \u003cp\u003eHypoxia vs. 60\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-5.638\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 114px;\"\u003e\n \u003cp\u003e-7.860 to -3.417\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.0009\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, our study provides the first known, direct quantitation of oxygen equilibration and reoxygenation dynamics in embryo culture media using conditions designed to mimic routine ART laboratory practices. We demonstrated that oxygen exchange is gradual but predictable with equilibration under hypoxia. Our results also demonstrate that reoxygenation can occur rapidly with significant rises in O₂ within minutes of exposure to ambient air. Additionally, the choice of oil overlay significantly alters these dynamics, with light oil allowing faster equilibration and reoxygenation compared to heavy oil. The influence of oil type impacts buffering capacity and reoxygenation rates, creating a dilemma in oil choice for embryo culture.\u003c/p\u003e \u003cp\u003eThe intent of our experimental design was to closely reflect practices in embryology laboratories. During routine \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) procedures, media preparation is frequently performed on the benchtop in atmospheric oxygen, after which plates are transferred into low oxygen incubators to more closely mimic the physiologic environment \u003cem\u003ein utero\u003c/em\u003e. The first portion of our experiment closely emulated this, as the dishes were prepared on the bench top then moved to a hypoxic incubator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, oocytes and embryos are often removed from the hypoxic incubator for micromanipulation steps such as intracytoplasmic sperm injection (ICSI) or embryo biopsy, briefly exposing them to atmospheric oxygen before being returned to reduced oxygen [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since embryo manipulation often takes place in atmospheric oxygen, to recreate the laboratory environment, the O2 sensor was equilibrated in the destination incubator, dishes were then transferred to a conventional 5% CO2, 37\u0026deg;C incubator and allowed to equilibrate overnight to atmospheric conditions to measure reoxygenation dynamics of embryo culture media (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSome ART labs perform their dish preparation in a preequilibrated incubator (18\u0026ndash;19% O\u003csub\u003e2\u003c/sub\u003e ) then moved into a hypoxic incubator. Therefore, we assessed hypoxia equilibrium when embryo culture media was moved from an atmospheric incubator to a hypoxia chamber (5% O\u003csub\u003e2\u003c/sub\u003e). In our experiment the dishes were then returned to the hypoxia chamber for at least 12-hours and then reoxygenated to mimic the routine handling steps, allowing us to quantify the equilibration dynamics of culture media under realistic laboratory conditions.\u003c/p\u003e \u003cp\u003eIn our study we used a hypoxic glove box incubator (hypoxia chamber) maintained at 5% O\u003csub\u003e2\u003c/sub\u003e which prevented reoxygenation artifacts during the process of manipulating the oxygen probe. This chamber also maintained constant temperature, which can also influence oxygen sensor readings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A primary goal of our study was to mimic culture conditions without inserting the additional influence of embryo metabolism. Our work highlights how fluctuations in environmental oxygen saturation could influence the stability of the microenvironment surrounding embryos. By characterizing the kinetics of oxygen equilibration across different manipulations, our study underscores the potential for transient reoxygenation events to affect embryo culture outcomes. This provides valuable insight into best practices for minimizing oxygen fluctuations, reinforcing the importance of consistent hypoxic culture conditions in optimizing IVF success rates.\u003c/p\u003e \u003cp\u003eBoth the light and heavy oil equilibrations followed single-phase decay curves, but the half-life to equilibration in light oil was significantly lower than the half-life for the heavy oil. These results indicate that longer equilibration times should be considered in standard laboratory workflows that use heavy oil. This highlights the impact of oil viscosity and density on oxygen diffusion, which had been suggested in theory but here is directly measured [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe result of our study has relevant implications for ART practice. Notably, oxygen concentrations used for embryo culture vary considerably across clinics and regions. Many laboratories continue to culture embryos at atmospheric oxygen (20%) despite recommendations supporting reduced oxygen conditions (5%) to more closely reflect the physiologic reproductive environment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Culturing embryos at elevated oxygen has been shown to cause massive gene deregulation in mice, including genes required for cell growth and gastrulation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, bovine embryos cultured under elevated oxygen exhibit increased DNA methylation and reduced blastocyst formation rates [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Exposure to atmospheric oxygen further enhances the activity of oxygen-dependent enzymes, leading to accelerated ROS generation, heightened oxidative stress, and prolonged embryo development [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This dynamic is particularly concerning given the known sensitivity of preimplantation embryos to oxygen fluctuations and the potential for reactive oxygen species (ROS) mediated damage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. ROS are often generated \u003cem\u003ein vitro\u003c/em\u003e and rise if oxygen tensions of the media increases, impairing embryo competence and viability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This can impair embryo competence and viability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study has some limitations. First, our kinetic data was derived from a media only system without the influence of embryo metabolism. Embryos may alter oxygen dynamics due to metabolic waste production and cellular oxygen consumption which may accelerate or delay equilibration rates [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Investigating oxygen kinetics in the presence of embryos, as well as directly measuring ROS levels, could provide more biologically relevant insights. Furthermore, only one formulation of culture media was tested therefore this data may not be generalizable for all other media types. Further studies would be recommended to compare multiple media types. Additionally, media droplets were pipetted into the dish to mimic standard embryo culture conditions, but only one of the droplets could be measured at one time due the oxygen monitor only having one sensor input. Attempts to measure changes in all four droplets in sequence resulted in equilibration artifacts due to the transition of the sensor between droplets. More consistent readings were achieved by fixing the probe in one droplet with a micromanipulator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Incubator space also did not allow for more than one micromanipulator per experiment. Finally, our study did not evaluate the impact of the shift in oxygenation on the embryo itself. It is not yet known if short term shifts in oxygen have an impact on embryo quality.\u003c/p\u003e \u003cp\u003eTaken together, these results argue strongly for minimizing atmospheric exposure during all phases of embryo culture and for implementing hypoxic workstations or isolettes wherever feasible. Moreover, our direct measurements of equilibration kinetics offer embryologists practical guidance on how quickly reoxygenation occurs and how oil overlay choices modulate this process. By defining these parameters, our study provides practical and quantitative benchmarks for reoxygenation kinetics, that contribute to the development of evidence-based best practices aimed at reducing oxygen-related artifacts, safeguarding embryo development, and ultimately improving IVF outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. The experiment was conducted by Adam Krieg and Thomas O\u0026rsquo;Leary. Data analysis was performed by Adam Krieg and Sanjana Kulkarni. The first draft of the manuscript was written by Sanjana Kulkarni and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge support from the National Institutes of Health R21HD094983 (AJK) and R01HD082208 (AJK) for this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFischer, B. and B.D. Bavister, \u003cem\u003eOxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits.\u003c/em\u003e J Reprod Fertil, 1993. \u003cstrong\u003e99\u003c/strong\u003e(2): p. 673-9.\u003c/li\u003e\n\u003cli\u003eMaas, D.H., B.T. Storey, and L. Mastroianni, Jr., \u003cem\u003eOxygen tension in the oviduct of the rhesus monkey (Macaca mulatta).\u003c/em\u003e Fertil Steril, 1976. \u003cstrong\u003e27\u003c/strong\u003e(11): p. 1312-7.\u003c/li\u003e\n\u003cli\u003eSciorio, R. and G.D. 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CD008950.\u003c/li\u003e\n\u003cli\u003eBedaiwy, M.A., et al., \u003cem\u003eDifferential growth of human embryos in vitro: role of reactive oxygen species.\u003c/em\u003e Fertil Steril, 2004. \u003cstrong\u003e82\u003c/strong\u003e(3): p. 593-600.\u003c/li\u003e\n\u003cli\u003eCatt, J.W. and M. 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Van Raamsdonk, \u003cem\u003eBeneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies.\u003c/em\u003e Front Cell Dev Biol, 2021. \u003cstrong\u003e9\u003c/strong\u003e: p. 628157.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"ART Media Preparation, Reactive Oxygen Species, Oxygen Saturation Equilibration, Media Equilibration","lastPublishedDoi":"10.21203/rs.3.rs-8143184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8143184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eOptimal oxygen tension is essential for successful \u003cem\u003ein vitro\u003c/em\u003e embryo culture in assisted reproductive technology (ART). Reduced oxygen levels (3\u0026ndash;8%) improve embryo development by minimizing oxidative stress, however, limited knowledge exists about transient oxygen fluctuations during handling outside hypoxic incubators. This study aimed to quantify the kinetics of oxygen equilibration in embryo culture media under conditions designed to mimic common ART laboratory practices.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEmbryo culture media droplets were prepared in room air and overlayed with light or heavy mineral oil. Dishes were equilibrated in a hypoxia chamber (5% O₂, 5% CO₂, 37\u0026deg;C), then transferred to an atmospheric incubator (18\u0026ndash;19% O₂, 5% CO₂) for equilibration, and then this was repeated once more. Oxygen saturation was measured every 30 seconds using a fiber optic microprobe (PreSens, GmbH). Each experiment was replicated three times, yielding six equilibration and six reoxygenation curves. Data were fit to single-phase exponential models to calculate half-lives and rate constants.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMedia equilibrated from atmospheric to hypoxic conditions within 12 hours. Light oil overlays demonstrated faster equilibration (half-life 71 min) compared to heavy oil (half-life 116 min, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Upon reoxygenation, oxygen saturation rose rapidly, with light oil droplets reoxygenating with a doubling time of 50 min and heavy oil in 78 min (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn ART media, hypoxic oxygen equilibration is a gradual process while reoxygenation is rapid. Oil viscosity significantly influences oxygen equilibration dynamics, with light oil permitting faster equilibration and reoxygenation. These findings underscore the importance of minimizing atmospheric exposure during routine handling and highlight the role of overlay oil in reducing transient oxygen fluctuations.\u003c/p\u003e","manuscriptTitle":"Oxygen Equilibration Dynamics in Assisted Reproductive Technology Embryo Culture Media","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 13:37:50","doi":"10.21203/rs.3.rs-8143184/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"31290996953821216404515828685641064603","date":"2025-12-14T22:32:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-10T21:56:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-22T20:18:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T04:51:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2025-11-18T08:40:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"074a1f17-db91-4150-a683-508cce184c87","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T12:56:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 13:37:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8143184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8143184","identity":"rs-8143184","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}