Cytokine profiling is a valuable tool for monitoring immune responses associated with disease and treatment. This study assessed the impact of sex and sedation on serum cytokines in healthy nonhuman primates (NHPs). Twenty-three cytokines were measured from serum using a bead-based multiplex assay. Assay validation for precision, sensitivity, recovery, linearity, and stability was performed. Samples from male and female cynomolgus and rhesus macaques either cooperating or sedated were compared. All cytokines except TNFα demonstrated acceptable sensitivity and precision, with variable recovery and linearity. IFNγ, IL-2, IL-5, IL-6, IL-8, IL-12/23 (p40), IL-13, IL-15, MCP-1, TGFα, VEGF met acceptance criteria; G-CSF, IL-4, IL-10, MIP1α, sCD40L were marginal. Higher cytokine levels were observed in females and cytokine levels were blunted in sedated NHPs when compared to awake cooperating NHPs. Significant differences observed in cytokines related to sex, species, or imposed by handling highlight the importance of model design on translational relevance for clinical settings. The release of pro-inflammatory cytokines, classically IL-1, IL-6, and TNFα, to combat infection has a profound secondary effect on the overall physiologic system that can result in hypotension and tissue damage, as is seen in sepsis2. Additionally, recent studies have demonstrated a similar pro-inflammatory cytokine storm leading to ARDS after infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic3. Interestingly, sedatives and anesthetics commonly used in critically ill and septic patients, as well as in surgical settings, have an anti-inflammatory effect thought to be due to a global reduction in pro-inflammatory cytokines and nitric oxide production5. This sedation effect may be beneficial in the treatment of sepsis, but also may drastically impact the interpretation of the immune response in cytokine-based studies where sedated or chemically restrained subjects are evaluated. This underappreciated impact of sedation and anesthetics on the immune system is important to consider when designing and evaluating scientific studies of changes in cytokine levels.Cytokines including chemokines, interferons, interleukins, growth factors, and tumor necrosis factors are mediators that play a critical regulatory role in inflammation and immunity. They exert their effects in shaping immunopathologic processes after secretion from primary cells by membrane-bound direct intercellular signaling. Cytokines are commonly used as important indicators of health, inflammation, and immune status. Since the discovery of the first interferon in 19576. Immune profiling cytokines in health and disease has led to the development of numerous immunotherapy medications, both agonists and antagonists, for many diseases such as adult and juvenile rheumatoid arthritis, psoriasis, Inflammatory Bowel Disease, melanoma, colon cancer, lung cancer, renal cell carcinoma, multiple myeloma, and AIDS. For example, Adalimumab, an anti-TNFα antibody, has been used successfully to treat Inflammatory Bowel Disease7. IL-2 and IFNα in combination with chemotherapy have been used to treat metastatic melanoma, but development of many other cytokine treatments like IL-15 as single agents has not proceeded beyond Phase 2 trials8. Clinical inefficacy or negative side effects have so far limited the wide clinical success of cytokine-based therapies10, which is related to cytokine signaling across multiple pathways and many that exhibit redundancy and pleiotropy. The complexity of these targets drives the need for accurate and reliable measurements of cytokines to continue to advance the understanding of immune pathophysiology and immunomodulation strategies.Cytokines transmit intracellular signals that are central to immune homeostasis or dysregulation and have a central role in pathologies like obesity and diabetes or cancer and autoimmunity. Conversely, the normal cytokine response can also be a target for immune manipulation like in the situation of transplantation where the host immune response to the graft must be suppressed to prevent rejection. In transplantation the achievement of immune tolerance reduces the risk of rejection and improves safety by eliminating toxic immunosuppression12 and educated immune system similar to that of humans makes the NHP a uniquely relevant model system that allows for valid modeling of human diseases, as well as for safety and efficacy studies of drugs, and implantable devices14. Humans and old-world primates, a group which includes cynomolgus and rhesus macaques, diverged from a common ancestor approximately 25 million years ago15 and macaques share 93% of their genome with humans16. One such example includes the genetic similarity between NHPs and humans in immunologically important MHC class I and II regions15. Additionally, the expression of Toll-Like Receptors within subsets of dendritic cells are analogous in humans and NHPs but not rodents, which has far-reaching implications for innate immune function13. In immune senescence research, a hallmark loss of CD28 expression by T cells in humans is accurately modeled by NHPs but not by rodents13. When using NHPs in preclinical testing, accurate, reproducible, and independently validated measurements of longitudinal and clinically relevant endpoints, such as levels of cytokines in serum, are critical. Unfortunately, validation reports supporting the reliability of methods for the measurement of cytokines in NHP serum are rare, and those that have been published do not have uniform acceptance criteria and validation methods19, making interpretation between studies challenging.Commonly used animal models such as the mouse have high value as screening or mechanistic models due to the wealth of information that can be obtained from genetic modifications and the availability of reagents. Nonhuman primates (NHPs) are used to bridge gaps that cannot be addressed by other animal models and because of high predictive validity are among the most robust translational model for infectious disease, neuroscience, transplantation, and reproductive biology in humans. The outbred nature of NHPs as well as anatomy20. Unfortunately, with the rapid improvements in cytokine multiplexing technology, the assays in this study are no longer available. Additionally, this study did not report intra- or inter-assay variability, which can play a large role in the quantitative results obtained.Multiplexed cytokine assays have gained great popularity in both human and animal research due to their ability to provide a more complete picture of the immune response, which is important given the complex interactions of the cytokine network in both disease progression as well as regulation of disease. Most assays in current use are similar to a traditional ELISA, but with each capture antibody coated onto individual beads which are then combined for the assay, allowing for the measurements of double-digit numbers of cytokines. Multiplex assays have the comparative advantage of small sample volume relative to the circulating blood volume in the NHP, making frequent serial sampling possible without compromising welfare to investigate disease progression from the subtleties to potential major biomarkers. While many studies report cytokine measurements from NHP blood using multiplex assays, there is a lack of method validation for multiplex cytokine assays in NHP model in the current literature. Method validation is necessary to account for any inherent methodological error and study limitations, and is critical to appropriately interpret reported values from the assay. Method validation procedures include the demonstration of analytical sensitivity, linearity, precision (intra- and inter-assay), and accuracy . Multiplex assays pose a unique challenge for validation given the myriad of possibilities for interference and cross-reactivity of so many analytes as well as the technical challenges of a complex bead-based assay. In 2014, He et al. described the only known validation of a multiplex cytokine assay in NHPs21. We evaluated cytokine secretion patterns in two NHP species  commonly used as models of infectious disease, neurodegenerative disorders, and regenerative medicine. Understanding and being able to reliably measure cytokine levels in healthy NHPs as a translational animal model is critical for future research into cytokine effects and roles in disease, allowing for the development of more personalized, robust, and effective therapeutics.We sought to independently validate a commonly-used, commercially-available multiplex cytokine assay for NHPs by establishing acceptance criteria for cytokine measurements by determining which cytokines in the assay met said criteria to ensure experimental rigor and support the proper evaluation of nonclinical laboratory studies subject to FDA, EMA, and ICH guidance. Once validated, cytokine concentrations were measured in serum from male and female sedated and cooperating cynomolgus and rhesus macaques. To determine if these sex and model handling variables are associated with baseline secretory patterns, we collected laboratory data retrospectively after the introduction of the validated assay. While such observations have been demonstrated in other species, they have not previously been made in macaques, a highly relevant animal model for preclinical testing. This is of interest because often a change from baseline levels or the balance between pro- and anti-inflammatory cytokines is what is associated with disease progression or improvementAll animal procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee, conducted in compliance with the Animal Welfare Act, adhered to principles stated in the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no 85-23), and studies performed and reported in compliance with the ARRIVE guidelines.Macaca fascicularis)  and 26 healthy rhesus macaques (Macaca mulatta)  were evaluated in this study . Animals were sedated with either 8–10 mg/kg ketamine (IM) (n = 27) or 4 mg/kg tiletamine combined with zolazepam (IM) (n = 2). The tiletamine/zolazepam-sedated animals were excluded from analyses comparing cytokine expression in cooperating and sedated NHPs. Of the 27 animals, 5 were sedated with ketamine only, and the remaining 22 were sedated with ketamine only (n = 11) or ketamine in combination with 0.1–0.3 mg/kg midazolam (n = 11) for anesthesia induction with inhalation isoflurane  for CBC evaluation, and serum separator tubes (0.6 mL) for chemistry evaluation.At time of sampling, cynomolgus macaques were aged between 3.0 and 12.9 years , and weighed between 3.11 and 7.94 kg . Rhesus macaques were aged between 1.7 and 13.1 years , and weighed between 3.05 and 16.2 kg . Blood samples were collected from awake, cooperating animals  via indwelling vascular access port25. Change in kit number was due to manufacturer transition in kit format from polystyrene to magnetic beads. All validation-related measures were performed using the magnetic bead-based kit. These kits includes premixed beads for 23 cytokines: G-CSG, GM-CSF, IFNγ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17A, MCP-1, MIP-1α, MIP-1β, sCD40L, TGFα, TNFα, VEGF and IL-18. Standard curves for each cytokine were prepared by serial dilution and run in triplicate . The standards and controls for nine of the cytokines  were recombinant NHP proteins; the standards and controls for the other 14 cytokines were human proteins. The detecting antibodies for IL-2 were generated against monkey (unspecified species) protein; the detecting antibodies for VEGF were generated against mouse protein; the detecting antibodies for the remaining 21 cytokines were generated against human proteins. The manufacturer reports “no or negligible” cross-reactivity between any of the analytes of the assay panel. A human serum matrix provided with each assay kit is added to the standard and control wells to mimic the NHP matrix of the samples. A Luminex100 system equipped with xPONENT v. 3.1 software was used to perform the multiplexed assays.Millipore Milliplex MAP premixed bead-based 23-plex kits for Non-Human Primate Cytokines  were used according to manufacturer instructionsSamples from 20 cynomolgus macaques were pre-selected as naïve and required sampling for other enrolled studies, allowing for opportunistic sampling for this study. These 20 animals were used for validation testing of the multiplex assay. All validation measurements were performed across four assay plates.To evaluate recovery (measured as observed concentration relative to expected concentration), eight unique serum pool samples were made from multiple samples taken from a single animal on different days, with the exception of one of the serum pools was constructed from samples of two different animals due to sample availability. Each sample was measured in three ways: neat (unspiked) and with spiking of a known amount of either of two manufacturer provided standards (highest concentration—kit “standard 7”—and mid-range concentration—kit “standard 5”). Each spiked sample consisted of 75% endogenous NHP serum and 25% kit standard  was evaluated using the same neat serum pool samples used in for the evaluation of assay recovery. Each sample was spiked with the highest concentration kit standard and then measured at 1:2 and 1:5 assay buffer dilutions. Three of the eight samples were also measured neat (unspiked) and at a 1:2 dilution  or high-concentration (kit “standard 7”) kit-provided standard to determine precision at limits. Each spiked sample was constructed such that > 90% of the final spiked matrix consisted of endogenous NHP serum. Each sample was tested with 12 replicates within a single assay plate  was measured at 1:2 and 1:4 assay buffer dilutions to extend each analyte’s standard curve. Both the lower limit of detection  and lower limit of quantification  were determined for each cytokine, though only the LLOD was used to determine if validation acceptance criteria were met (LLOD ≤ the lowest standard). Each dilution was tested on a single assay plate six times , three serum samples, each taken from a different cooperating cynomolgus macaque were measured neat (unspiked), and spiked with ketamine. Ketamine was diluted to 0.02 mg/mL with kit-provided sample matrix, and added to serum samples to give a final concentration of 2000 ng/mL, the reported peak concentration of ketamine in human plasma during sedationCytokine concentration was determined by fluorescence intensity. Fluorescence data were analyzed with Millipore Milliplex Analyst version 3.4 according to manufacturer recommendations. All statistical analyses were performed with the calculated concentrations of each sample rather than the MFI (mean fluorescence intensity) source data to give a more accurate representation of assay performance.Assessment of intra- and inter-assay precision was measured by the coefficient of variation (CV%) calculated from the measured concentration of each replicate. Measurements of sensitivity, recovery, and linearity were calculated as a ratio of the assay returned cytokine value compared to the expected cytokine value.27. Significance between two NHP groups was determined using non-parametric unpaired, two-way Mann–Whitney tests with α = 0.05. Significance between the neat and ketamine-spiked serums samples was determined using non-parametric Wilcoxon matched pairs signed rank tests with α = 0.05. Multivariate analysis was conducted using a two-way ANOVA with Box-Cox transformed data to meet statistical assumptions. The two-way ANOVA table was generated using software from Assaad et al.28. All data sets were tested for normality using the Kolmogorov–Smirnov test, and unequal variance was assessed using the F-test.Statistical analysis was performed using GraphPad Prism version 8.4.1 . Outliers were identified using the Tukey method but were not excluded. Data points falling below the LLOD were adjusted to half the value of the LLOD for that cytokine (i.e. < 0.64 was changed to 0.32)Final recovery acceptance criteria for a given cytokine was defined as more than half of all samples for a given analyte meeting the acceptance criteria of 75–125% of the expected value. Thirteen of 23 cytokines met validation acceptance criteria for recovery , recovery performance varied considerably across the samples. For example, in one assay, TGFα showed an average recovery of 114% in one animal, but an average recovery of 70% in another. In contrast, three analytes  were consistently recovered across animals and assays—each had acceptable recovery levels across all determinations. GM-CSF consistently had a recovery of between 200 and 300%. Four analytes  showed consistently low recoveries across animals and assays  of endogenous (neat) samples) Fig. . All thr29). All cytokines met acceptance criteria of CV < 20% for intra-assay precision  met the acceptance criteria for inter-assay precision. Validation acceptance criteria was more varied for recovery and linearity. Final recovery and linearity acceptance classification for each cytokine are summarized in Table Each of the 23 cytokines were categorized as acceptable, marginal, or rejected based upon validation acceptance criteria for sensitivity, precision, recovery, and linearityDetectable cytokine ranges were determined for male and female cohorts of both cynomolgus and rhesus macaques Table ; these cTo assess the effect of sedation on cytokine detection, detectable ranges were also determined for sampling-method subgroups (cooperating vs. sedated) cynomolgus macaques Fig. ; rhesus To further investigate the interaction between sex and sedation on cytokine expression, we compared the cytokine expression of each cohort subset—cooperating females, sedated females, cooperating males, and sedated males Table . We founOf the 23 cytokines that underwent validation in healthy NHP serum, 11 met all acceptance criteria, 5 were marginally accepted, and 7 were rejected. These results demonstrate that the Millipore Milliplex multiplex assay can be reliably used to detect each of the 11 accepted cytokines  and VEGF) at endogenous levels in serum from healthy NHPs. The marginal cytokines meeting most of the validation criteria  require consideration of method specific limitations prior to interpretation of absolute measurements. The 7 cytokines in the panel that did not meet our pre-established acceptance criteria  may not be reliably detected using this method in healthy NHP serum. This validation was performed entirely on frozen samples as is typical with batch assays; further testing to evaluate validation in fresh samples or samples undergoing freeze–thaw cycles are needed. Rejected cytokines were excluded from future analyses in this study since results were deemed invalid.This study confirms this multiplex assay is capable of detecting specific cytokine concentrations in healthy male and female cynomolgus and rhesus macaques both consciously cooperating and sedated for other procedures and calls attention to the importance of cohort and model-handling experimental parameters when evaluating cytokine levels. We demonstrate significant differences in male vs. female and cooperating vs. sedated cynomolgus macaques, as well as baseline differences between cynomolgus and rhesus macaques. Interestingly, we saw an overall reduction in, or blunting of, cytokine levels after sedation of both male and female cynomolgus macaques. We confirmed that this was due to a physiologic decrease in cytokine levels as opposed to direct assay interference with ketamine by spiking ketamine in vitro into the samples, showing no difference in cytokine levels between unspiked and spiked samples. Of note, cytokine levels were measured immediately upon/during sedation, and evaluation of the short- and long-term effects of sedation will be critical in understanding implications.The use of a retrospective study design allowed us to aggregate a large NHP data set that is an advantage over the small experimental groups generally used in NHP modeling, however, this approach introduces inherent limitations. We observed wide inter-animal variation in cytokine expression, and while day-to-day within-subject (intra-)animal variability is relatively stable compared to population variability, subsequent follow-up studies could address this in combination with other factors prospectively. Even considering this variability, sedation was still significantly associated with cytokine blunting, suggesting subsequent studies are worthwhile to study the specific impact of each unique regimen on cytokine levels and is intentionally balanced for demographic factors which could not be done retrospectively with this cohort.33. The blunted cytokine response we demonstrated does not seem to be pathway-specific, as both IL-12/23 (p40) (a potent Th1 polarizing cytokine) and IL-10 (a potent Th2/regulatory cytokine) are reduced after sedation, which highlights the importance of understanding sedation effects in various disease states given the dual effects of Th1- or Th2-driven pathways. To our knowledge, the effects of sedation on cytokine levels in NHPs has not been investigated or described previously. Anesthetics and sedatives such as propofol, dexmedetomidine, and ketamine, which are commonly used in the intensive care setting for the purposes of sedation and pain control for intubated and/or critically ill patients, have also been shown to reduce peripheral cytokine production in humans and rodents, consistent with our findings. Specifically, Propofol and ketamine reduce the production of pro-inflammatory cytokines TNF-a, IL-6, and IL-8 in LPS-challenged in vitro cells as well as in induced rodent models of sepsis34. Additionally, use of sedatives in rodent models of sepsis has shown improved outcomes such as end-organ dysfunction and mortality, which has been postulated to be due to a reduction in the overwhelming systemic inflammation of sepsis33. These drug combinations are commonly used in clinical settings both in intensive care and the perioperative period.This blunted cytokine response after sedation has profound implications in preclinical and clinical research and treatments. A blunted cytokine response could negatively impact clinical outcomes such as wound healing and the ability to overcome infection. However, it also has the possibility for positive impacts on clinical outcomes such as mitigating transplant rejection and reducing the cytokine storm that contributes to tissue damage and end-organ dysfunction in sepsis35. Sex-dependent differences in immune responses need to be tested in a more dedicated manner in the future in order to develop therapeutics that are effective for individuals of any sex. A recent study noted that within a sex-balanced study of healthy human serum, there was significant variation between expression levels in males and females, though this variation was not explained by the estrous cycle of female subjects36. This emphasizes the importance of designing studies with both baseline controls within subjects, as well as sex-balancing between subjects within a cohort.We additionally observed overall higher levels of both pro-inflammatory and anti-inflammatory cytokines in female cynomolgus macaques in comparison with male cohorts. These sex differences are especially critical to recognize as preclinical and even clinical studies are often male-skewed, which can dramatically impact efficacy of treatments for female patients. Studies investigating sex differences in the immune response have been sparse, though it is generally accepted that adult female mammals have greater inflammation at baseline than males consistent with our data38. This highlights the difficulty of determining a ‘reference range’ for each cytokine; instead, we advocate for using each animal’s healthy state as a baseline and assessing change over time in response to illness or treatment, a strategy proposed for disease monitoring in human patients. Our stability studies show an average threefold change in all cytokines for samples taken from the same healthy animal on different days. Similar trends and challenges are also seen in human studies. Inconsistencies in cytokine measurements have also been reported in human multiplex kits18, reporting variable recoveries, indicating that the challenges of measuring cytokines simultaneously in a single matrix are not specific to NHPs. Our findings are similar to those from studies of the stability of endogenous cytokine expression in humans over time, which found a greater variability in cytokine expression between individuals at the same time point, than there was from the same individual across time points. In fact, NHPs are an ideal preclinical model as they mimic the heterogeneity, both inter- and intra-animal, that is seen in human cohorts.In addition to the differences seen between cohorts of sex and sedation status, our data shows a high level of variability between individual animals as demonstrated by large standard deviations despite relatively high sample numbers. Such variability, despite being exposed to similar environmental conditions, may be attributed in part to the outbred nature of primates. Similarly high levels of cytokine variability have also been recognized in other species, including dogs and humans39, and exhibit dynamic expression patterns that are independent of illness. Expression of various matrix proteins (including blood clotting factors and hormones) have been shown to exhibit diurnal and seasonal fluctuations in humans41. Such fluctuations may account for the intra- and inter-animal variability observed in this study. Diurnal fluctuations of cortisol have been shown to have an inverse effect on the expression of several inflammatory cytokines . Humans have lower levels of cortisol expression between the hours of midnight and 5:00 a.m., which may contribute to noted increases in inflammatory symptoms at night42. This demonstrated relationship between cortisol and inflammatory cytokines suggests that an increase in cortisol as a response to stress may directly influence the expression of inflammatory cytokines, making this relationship critical to the interpretation of inflammatory markers in animal subjects undergoing handling related stresses.Interpretation of cytokine levels, either individually or in a multiplex assay, has many inherent challenges. Cytokines are, by nature, pleiotropic in their effects43. For example, in one study of neonatal sepsis, cutoffs for IL-6, TNFα, and IL-1β were found to facilitate diagnosis on day one of infection, and combining these multiple cytokines enhanced accuracy in diagnosis, suggesting that all 3 cytokine levels in relation to each other are important in disease pathogenesis. However, these cutoff levels even in well-studied diseases such as sepsis have not yet been implemented routinely in the clinical setting, as additional work is needed to support their utility across populations and also to harmonize test methods44. Further, cytokine expression has been shown in humans to be influenced by a variety of other continuously changing demographic characteristics, including both downward (e.g. IL-2Rα) and upward (e.g. IL-17) changes in expression with age45. The cohorts used in this study displayed minimal age-related differences in cytokine expression, though this finding is likely explained by an age distribution skewing heavily towards the 4–6 years age range with only a small number of older animals. This range reflects the modeling of young healthy adults, but evidently ongoing studies would benefit from including a cohort of older, adult animals to better explore the effects of age on cytokine expression in macaques especially in studies where animals across the age spectrum are of interest .While we present absolute cytokine levels, it should be mentioned that interpretation requires context because “normal” values have not been well established for most cytokines. As cytokine levels vary by the individual and type of disease being studied, more commonly the balance between cytokines within the overall physiologic system or a change in levels from an individual’s baseline are associated with worsening or improving disease46. Furthermore, the need to evaluate multiple cytokine levels together to better understand the overall clinical picture of a subject makes multiplexing an attractive method. Comparisons of multiplex-based immunoassays with single analyte ELISAs showed that while measurements of high abundance cytokines were highly correlated between methodologies, low abundance cytokines in healthy human subjects  were poorly correlated47. This trade-off between multiplexing and assay sensitivity for low abundance cytokines must be carefully considered when designing a study, and caution should be used when interpreting results near the LLOD of a multiplex assay. The popularity of multiplex assays and the importance of NHPs in pre-clinical testing has resulted in an increase in the number of NHP multiplex cytokine assays on the market. Given the importance of understanding the cytokine response in pre-clinical testing, it will be critical to continue validation efforts of these commonly used assays.Multiplexing technology has significant animal welfare and research advantages when compared to traditional methods like ELISA; the ability to simultaneously measure multiple cytokines from a single small volume sample allows for reduced animal handling, reduced blood draw volume, and increased research efficiency. While multiplex assays cost more than individual ELISAs, they are relatively cost-effective when four or more analytes can be accurately measured48, making changes in expression of low-abundance cytokines harder to detect in serum. Differences in the composition of the sample matrix, including the protein-rich environment of biological fluids, may lead to cross-reactivity within the assay, resulting in poor analyte recovery such as was noted in this study . For example, a common plasma protein, alpha-2 macroglobulin, has been shown to interfere in ELISA assays and lower the detection of IL-2, IL-6, IL-4, IL-1β and TNFα by up to 26%49. Additionally, autoantibodies can inhibit their cytokines in vitro, leading to artificially low measurements of the targeted analytes50. Conversely, artificially high cytokine measurements can result from soluble cytokine receptors being present in the blood matrix51. For these reasons, it is critical to consider sample matrix when designing multiplex experiments.The sample matrix (serum or plasma) can affect the detection of circulating cytokines, specifically those that are expressed near the limit of detection. Non-specific background is significantly increased in serum as compared to plasmaThis work demonstrates uniqueness in individual cytokine profiles and the influence of sex, species, and sedation. The NHP provides an ideal model in which to study these questions as both consciously cooperating and sedated animals of both sexes can be sampled, and healthy animals can be compared to a multitude of disease processes of interest. This provides a stepping stone for future work in evaluating population- or subject-based reference intervals for cynomolgus and rhesus macaques. Specifically, understanding the role and mechanisms by which anesthetics and sedatives reduce inflammation will help improve patient outcomes, especially in infectious processes such as sepsis and ARDS from COVID-19.Multiplexing cytokine measurements is an important research technique that improves efficiency, preserves biological samples allowing for reduction in number of animal subjects needed, and gives a wealth of complex information about the clinical state of a subject. This study outlines the limitations and challenges involved in detection and interpretation of multiple cytokines in healthy NHP serum, as well as the variability in cytokine expression due to species, sex, and sedation. Of the 23 cytokines validated in healthy NHP serum for the Millipore Milliplex assay, 11 met all acceptance criteria  and VEGF). Five were marginally accepted as they didn’t meet acceptance criteria for either recovery or linearity . Seven were rejected as they didn’t meet acceptance criteria for both recovery and linearity , indicating that these analytes may not be reliably detected using this method in healthy NHP serum.Prior to interpretation of raw multiplex cytokine data, it is critical to identify potential influences on cytokine expression, including species, sex, sedation, stress, and collection method. Following sampling, method-specific limitations to consider include matrix effects, species specific binding, and potential interactions between analytes that are included in a multiplex assay. Validation studies should be undertaken to account for these influences, and subject cohorts should be thoughtfully designed in a sex-balanced manner and with attention to model handling aspects such as sedation to ensure accurate interpretation and reporting of results from multiplex cytokine assays. In our study, we demonstrate the detectable levels and mean levels for cohorts of cynomolgus and rhesus macaques. We also demonstrate overall higher levels in female vs. male cynomolgus macaques and cooperating/awake vs. sedated cynomolgus macaques, which has possible significant clinical implications such as in transplantation and infectious disease modeling.Supplementary Information.


Information on birth outcomes were obtained from birth records. Maternal sociodemographic and lifestyle factors were obtained from questionnaires. We used linear regression models for continuous outcomes , and birth length (BL) and logistic regression models for binary outcomes (preterm birth (PTB), term low birth weight (TLBW) and small for gestational age (SGA)). Sensitivity analysis was performed using multiple imputation for missing covariate data.Trimester and source-specific PM10 from traffic were associated with 17 to 18% increased odds of TLBW in all pregnancy periods. We also found odds of TLBW increased by 40%  and odds of SGA increased by 18%  per IQR (6.54 μg/m3) increase of total PM10 exposure in the third trimester.After adjustment, interquartile range increases in source specific PM10 exposures affect birth weight, with particular concern in relation to exposures to PM10 from road transport sources; results for total PM10 suggest greatest effect in the third trimester. Effect size estimates relate to exposures in the 1990s and are higher than those for recent studies – this may relate to reduced exposure misclassification through use of full residential history information, changes in air pollution toxicity over time and/or residual confounding.This study adds to evidence that maternal PMThe online version contains supplementary material available at 10.1186/s12940-020-00684-w. Maternal exposure to particulate matter (PM) may affect fetal growth, resulting in adverse birth outcomes, including infant death, stillbirth, preterm birth (PTB), term low birth weight (TLBW) and small for gestational age (SGA) , 2. This10 by trimester, but results regarding potential sensitive time windows during pregnancy on adverse birth outcomes are inconsistent [2.5 sources or chemical components in relation to pregnancy outcomes [10 [There are several studies that have investigated effects of PMnsistent –11. Revinsistent , 12–16 houtcomes –22, feweomes [10 –25.This study used the Avon Longitudinal Study of Parents and Children (ALSPAC), with source-specific maternal air pollution exposure derived from dispersion models that distinguished between local and regional sources for all maternal residential addresses in pregnancy. The aim of this analysis was to investigate the associations between source-specific air pollution in each trimester of pregnancy and adverse birth outcomes, including birth weight, PTB, TLBW, SGA, head circumference (HC), and birth length (BL).The ALSPAC cohort is a population-based cohort study located in south-west England Fig.  a 26, 2, 29. TheAll pregnant women who lived in this area with an expected delivery date between 1 April 1991 and 31 December 1992 were eligible for the study . In totaEthical approval for this study was obtained from the ALSPAC Ethics and Law Committee and Local Research Ethics Committees.10 (referred to as PM10_road and PM10_other hereafter) within the study area. PM10_road was modelled from each main road, which have annual average daily traffic (AADT) > 5000, as a line source. PM10_other was modelled using meteorological data such as hourly values of wind speed, wind direction, cloud cover, and temperature obtained from British Atmospheric Data Centre (BADC) (www.badc.ac.uk) and information available on a 1 km grid from the National Atmospheric Emissions Inventory (NAEI) (http://naei.defra.gov.uk/data/mapping).A detailed description of the exposure assessment is presented in the paper of Gulliver et al. . In brie10 using meteorological data taken from the ERA-Interim meteorological reanalysis produced by European Centre for Medium-Range Weather Forecasts (ECMWF) and pollution data in the form of daily average concentrations of PM10 for 14 receptor locations. Total short-term PM10 , were evaluated using Eq. .The NAME-III air pollution model  was used to estimate regional/long-range sources (i.e. outside the study area) of daily PM10 air pollution monitoring in the area; from January 1993 until September 2005 there was a single site. In the absence of enough monitoring data to evaluate the model for periods relating to pregnancy trimesters, we validated the model using weekly averaged measured concentrations from 1993 [For the period of our study, monitoring in the Bristol area varied, but remained limited: from 1990 to 1992 there was no PMrom 1993 .10 exposure estimates in each trimester and the whole pregnancy (WP) [We assigned exposure estimates based on home address locations and averaged address-time-weighted PMncy (WP) , 31. TheMain outcome measures were derived by abstraction of maternal medical records by trained research midwives/nurses and included birth weight , HC , BL , PTB (binary), TLBW (binary), and SGA (binary) . PTB was2), maternal educational level (Low: O level or below/High: A level or above), maternal smoking during first 3 months of pregnancy (Yes/no), and environmental tobacco smoke exposure during pregnancy (Yes/no).The following potential confounders were identified a priori: infant’s sex , maternal age at 8 weeks of gestation (in years), pre-pregnancy maternal BMI  with birth outcomes, jointly adjusted for the other two PM10_total TAEs . We additionally conducted some multiply adjusted analyses to fit PM10 road and PM10 other simultaneously to the models for each pregnancy period.We also tested for effect modification by sex, maternal education and gestational age of the associations between PM for all pregnancy period and the birth outcomes Effect modification by ethnicity was not computed in this study because only 2.2% of the 80% of women who answered the question at 8 months post-birth identified themselves as non-white ethnicity, representative of the area at that time. We also ran mutually-adjusted models to estimate associations of one PMThe length of pregnancy may affect our results. Therefore, for the TLBW and SGA analyses, we compared whole pregnancy exposures truncated to 37 weeks among term births to ensure equal length of exposures.mi impute mvn command. The following variables were imputed: maternal age, pre-pregnancy maternal BMI, maternal educational level, maternal smoking, and environmental tobacco smoke exposure during pregnancy. Exposure and outcome variable of each models were considered as observed covariates and used in the models to impute theses variables. For each imputation model, 10 imputations were run. We then fitted the desired model separately on each of the 10 imputed datasets and combine the results for all regression models.Given the proportion of missing data on confounder variables were high in the study population, analyses based on complete cases  may be biased. Therefore, we used multiple imputation (MI) analysis , 34. We All analyses were performed using STATA version 15.Table 10 exposure levels were 0.81 μg/m3 (IQR: 0.72) for PM10_road, 5.17 μg/m3 (IQR: 2.29) for PM10_other and 32.60 μg/m3 (IQR: 3.79) for PM10_total during the whole pregnancy period  . High correlations were also seen between trimesters for other sources (PM10_other) (Pearson’s r > 0.8). Correlations for PM10_road and PM10_other in the same pregnancy period were not highly correlated  and SGA ,. A small decreased risk between PTB and PM10_total was found in T2 . PM10_total exposure in T1 was found to be associated with very small increases in HC  and BL  after adjustment.In adjusted models Table  expresse10_road was associated with increased risk of TLBW in all pregnancy periods except T1.For road traffic source air pollution, we found PM10_other exposure in T1 was associated with very small increases in HC  and BL  after adjustment.We also found PM10_other in relation to birth weight but not TLBW. For an IQR increase in PM10_other in T3, associations for O level or below ; for A level or above (higher level of education): 28.84 g . For PM10_other in WP corresponding associations for O level or below were − 0.74 g, 95% CI: −19.78, 4.09; A level or above: 25.14 g, 95% CI:4.62, 45.65birth weight. We also found an interaction effect between maternal education and PM10_road in T1, T2, T3 and WP for HC   and on BL  .In PM10_total exposure in T3 was associated with a higher risk of TLBW and SGA . After restricting the analysis to first child, we found higher effect sizes for all PM measures with birth weight and these became formally statistically significant for PM10_other in T1 and WP. However, little change was seen in other outcomes. . We also observed stronger associations between PM10_road and TLBW in T1 that now reached statistical significance . Associations of PM10_road for T2, T3 and WP changed slightly with narrower confidence intervals. PM10_other effects on TLBW were found in all pregnancy periods except T1 (with 19% to 23% increased odds).In multiple imputation models Table , adverse10_total, and BL for PM10_other and PM10_total remained similar after imputation  was also associated with increased risk of both TLBW and SGA, but this was driven by exposures in the third trimester. We additionally found some evidence for very small increases in HC and BL with higher PM10 exposures.We analysed associations between source-specific PM10 in later stage of pregnancy is associated with maternal cardiovascular alterations in blood viscosity and coagulability [Results specific to T3 are biologically plausible since the third trimester is the period of greatest fetal weight gain –37 suppolability , 43. Thilability , 45.10 in T3 on TLBW in the 1990s are larger per unit mass PM10 than in a large study in London for 2006–10 based on birth registration data, which had lower concentrations of PM10  [10 units, Smith et al. found odds of TLBW increased 4%  per 10 μg/m3 increase in T3 PM10 exposure ; unlike our study, the ESCAPE analyses found little evidence of increased ORs for TLBW for T3 exposures [Our finding of negative effects of total PMxposures .10 exposure on birth weight.Compared with the ESCAPE and London studies , 46, our2.5 traffic exhaust on TLBW was 2% stronger than that of PM2.5 traffic non-exhaust  [10 or other pollutants with HC and weight at birth [10 air pollution with HC and BL. These are of unclear clinical importance and may be chance effects and are somewhat contradicted by the simultaneous finding of an increased risk of TLBW. We did not examine fetal loss in these analyses, but a possible explanation for this is that the most exposed fetuses who are more vulnerable might have been lost if exposed to higher air pollution exposure in early pregnancy.HC at birth is an important measurement because of the potential effects of air pollution on neurodevelopment . Few preat birth . We obse10 exposure. We note that a recent paper from Scotland by Clemens et al. looking at births between 2002 and 11, did not find associations between birth weight and PM10 [10 concentration estimates based on calendar year average at a spatial resolution of 1 km × 1 km for PM10 and assigned PM10 based on postcode of address at the time of birth, which may have introduced significant misclassification of exposure and potentially explain failure to detect associations with birth weight. However, we acknowledge that in our study, errors, gaps, and overlaps of address history may still result in some exposure misclassification. Further, as information on participant’s time-activity pattern was not available, exposure estimates in this study refer only to ambient concentrations at home addresses while other exposure concentrations of each activity space were not considered .A major strength of this study is that we had a full reconstructed residential history, accounting for residential mobility for each study cohort member using a cohort contact database  and wereand PM10 . The autA major limitation of this study is the missing data on some key confounding variables. We reported percentage of missing confounder variables before imputation Table  which inTo examine impact of missing confounders we conducted imputation analyses addressing this. Results from imputation analysis strengthened our main findings including larger effect sizes seen with birth weight, TLBW and SGA, with narrower confidence interval. Also, for all imputed models, we found a reasonable low fraction of missing information (FMI) < 0.53  , 56, mayFor PTBs, the third trimester is of very different lengths compared with term infants. To ensure equal length of exposures, we restricted whole-pregnancy exposures to 37 weeks to ensure equal length for TLBW and SGA, the results remained consistent  approximate date. Additionally, exposure during periods preceding the conception was not considered. This may lead to an over- or under-estimate of the length of gestation, an inaccurate exposure estimate in each trimester and subsequently it may result in effects of each trimester being larger than effects of the entire pregnancy period averaged .10 exposures on birth weight outcomes, with particular concern about exposures to PM10 from road transport sources and results for total PM10 point to greatest effect in the third trimester. Mitigation and prevention policies in ambient air pollution management are important to reduce the burden of TLBW and PTB, which have potential for lifelong impacts on morbidity and future mortality risks at the population level.This study supports a differential effect of maternal PMAdditional file 1.


Background: Reproductive outcomes after fresh in vitro fertilization/intracytoplasmic sperm injection–embryo transfer (IVF/ICSI–ET) cycles are diverse in infertile women with a history of ovarian cystectomy for endometriomas. We aimed to develop a logistic regression model based on patients' characteristics including number of embryos transferred and stimulation protocols to predict the live birth rate in fresh IVF/ICSI–ET cycles for such patients.Methods: We recruited 513 infertile women with a history of ovarian cystectomy for endometriomas who underwent their first fresh ET with different stimulation protocols following IVF/ICSI cycles in our unit from January 2014 to December 2018. One or two embryo are implanted. Clinical and laboratory parameters potentially affecting the live birth rate following fresh ET cycles were analyzed. Univariable and multivariable analyses were performed to assess the relationship between predictive factors and live birth rate.Results: The overall live birth rate was 240/513 (46.8%). Multivariable modified Poisson regression models showed that two factors were significantly lowers the probability of live birth: female age ≥ 5 years ; BMI range 21–24.99 kg/m2 compared with BMI <21 kg/m2 . And two factors significantly increased the probability of live birth: AFC >7 ; two embryos transferred .Conclusions: For these infertile women who had undergone ovarian cystectomy for endometriosis, female age <35 years, AFC > 7, and two embryos transferred might achieve better clinical fresh IVF/ICSI–ET outcomes. BMI <21 kg/m2 or ≥25 kg/m2 might also have positive effects on the live birth rate, but different ovarian stimulation protocols had no significant effects. However, a larger sample size may be needed for further study. Endometriosis reduces the cumulative live birth rate by reducing the number of embryos that can be transferred rather than their quality . A retroTherefore, the purpose of this study was to identify the predictors affecting the clinical outcomes of in fresh IVF/ICSI–ET cycles among infertile women who had undergone ovarian cystectomy for endometriomas. And to investigate whether different stimulation protocols influence the live birth rate. This may help fertility specialists and their patients to determine the expectations of appropriate treatment strategies for ovarian endometrioma before starting assisted reproductive treatment.This was a retrospective study using the records of infertile women with a history of ovarian cystectomy for endometriomas who underwent the first fresh ET cycle following IVF/ICSI treatment at the Reproductive Hospital Affiliated to Shandong University from January 2014 to December 2018. This original study was approved by the Institutional Review Board of the School of Medicine, Shandong University (No. 2019-1-14). In all, the records of 513 patients were collected, of whom 240 had achieved a live birth. Both laparoscopic and open techniques were used for cystectomy for endometriomas. The inclusion criteria included: (1) ovulatory women aged ≤ 40 years; (2) women with a history of surgery for unilateral or bilateral ovarian endometrioma(s); (3) women undergoing the first cycle of IVF/ICSI treatment and receiving a fresh ET; and (4) ovarian stimulation protocols including long/prolonged/short GnRH agonist protocols and a GnRH antagonist protocol. The exclusion criteria were the presence of the following: (1) uterine malformations; (2) untreated submucosal uterine fibroids or multiple uterine fibroids; (3) adenomyosis; (4) male or female chromosomal abnormalities; and (5) patients who had undergone cyst aspiration or oophorectomy.All participants were monitored and managed according to the hospital's clinical protocols. Doctors chose different ovarian stimulation protocols on the basis of long-term clinical experience, combined with the patient's age, ovarian function and other individual conditions. There were four stimulation regimens used: a long GnRH agonist protocol ; a prolonged GnRH agonist protocol ; a short GnRH agonist protocol ; or a GnRH antagonist protocol  . Doctorst-tests. Quantitative data with non-normal distribution are expressed as medians and (range) compared with the Wilcoxon signed-rank test. Categorical data are represented as frequencies and percentages; variables in these measures were compared between live birth and covariates using Pearson's chi-squared tests. A P < 0.05 was assumed to be significant. Variables included in logistic regression models were selected on the basis of those shown to have a relationship with the main outcomes as characterized by P < 0.10. Results are presented as odds ratio (OR) and confidence interval (CI). Backward stepwise selection was performed to determine independent covariates. A P-value of 0.05 was considered significant. Because the study was retrospective, patients were not randomly assigned to groups and there were no missing data to be considered. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the performance of the live birth rate prediction model. Forest plot were performed using Graphpad Prism .Statistical analysis was performed using IBM SPSS Statistics . Quantitative data with normal distribution are expressed as the mean ± standard deviation (SD) and were compared with independent-samples Student's 2 levels. In the non-live birth group, 155 women are primary infertility and 118 women are secondary infertility. In the live birth group, 137 women are primary infertility and 103 women are secondary infertility. Women with a successful ART cycle were younger . The live birth rate for woman with a BMI range of 21–24.99 kg/m2 was lower than in women with BMI <21 kg/m2 and BMI ≥ 25 kg/m2 (P = 0.003). There were no significant differences in the basal luteinizing hormone (LH) level between the groups without and with a live birth .In this study, the records of 513 IVF/ICSI procedures in patients who had undergone ovarian cystectomy for endometriomas met the criteria and were included in this analysis. There were 284 pregnancies (55.4%), of which 38 (7.4%) were followed by miscarriages and 6 (1.2%) were ectopic. There were 240 live births (46.8%). Patient characteristics are summarized in P = 0.013). We found that the live birth rate for women with an AFC > 7 was higher than in those with AFCs ≤ 7 (P = 0.004). The live birth rates were significantly higher with a start-up gonadotropin dosage <200 IU (P = 0.029), with cleavage stage embryos transferred (P = 0.032), and with two embryos transferred (P = 0.004). There were no statistically significant differences in live birth rates in terms of oocyte fertilization method, total gonadotropin dosage, endometrial thickness on the hCG trigger day, E2 level on the hCG trigger day, number of oocytes obtained, and the numbers of top-quality embryos transferred. There were only 28 patients with BMI <18.5 kg/m2 and 18 with BMI ≥30 kg/m2, and most of the women were concentrated in the range of 18.5–30 kg/m2. Thus, we could divide them into groups according to WHO criteria , but also divide each group into subgroups for analysis. Therefore, we analyzed the live birth rate for every unit of BMI, and observed a significant decrease in the live birth rate for women with a BMI of 21–24.99 kg/m2, so we divided the BMI into the following 3 groups: <21 kg/m2, 21–24.99 kg/m2, and ≥25 kg/m2. The dramatic decline in fertility for women aged ≥35 years is widely accepted as dogma in ART clinics. So we divide by age into ≥35 years and <35 years groups.We defined poor ovarian reserve as an AFC of ≤ 7 and normal ovarian reserve as an AFC of >7. In the original data, some values for the E2 level on the hCG trigger day were recorded as continuous variables, while some were recorded as >3,000 pg/mL, so we divided them into 3 groups: <2,000 pg/mL, 2,000–3,000 pg/mL, and ≥3,000 pg/mL. The cut-off values of gonadotropin treatment duration, start-up gonadotropin dosage, total gonadotropin dosage, and endometrial thickness on the hCG trigger day were all located near the mean or median, where they had the greatest statistical effects.The embryological and clinical factors that might have affected the live birth rate after ET are shown in P = 0.177, which indicated that the data fitted the model well. Female age, BMI, basal LH, AFC, ovarian stimulation protocol, start-up gonadotropin dosage, gonadotropin duration, stage of embryos transferred, number of embryos transferred were included in the logistic regression model. Multivariable modified Poisson regression models showed that two factors were significantly lowers the probability of live birth: female age ≥ 35 years ; BMI range 21–24.99 kg/m2 compared with BMI <21 kg/m2 . And two factors significantly increased the probability of live birth: AFC >7 ; two embryos transferred . The area under the curve (AUC) of the receiver operating characteristic (ROC) curve for the live birth rate prediction model was 0.757 (95% CI 0.610–0.740) indicating a fair performance. The forest plot shown in We performed univariate regression analysis of variables predicting the incidence of live birth, as shown in The main objective of our study was to assess the factors associated with live birth rate in infertile women who underwent their first IVF/ICSI–ET after cystectomy for ovarian endometriosis. In this single-center case-control study of the records of 513 patients, we found that female age, BMI, AFC, and number of embryos transferred were important factors that affected the live birth rate. When age was considered as an independent variable, the results showed that older patients (35 years old) had a lower live birth rate (23.8%). Multivariate analysis confirmed these findings and identified patient age as a factor independently related to live birth rates. Such conclusions are generally regarded as dogma by IVF procedures –28, seemU-shaped curve), rather than linear (a straight line). After multivariable analysis, the live birth rate was higher for women with a BMI ≤ 21 kg/m2 and BMI > 25 kg/m2 than for those in the BMI range of 21–24.99 kg/m2. This means that when we considered the impact of BMI on reproductive outcomes, we can not only divide them into groups according to WHO criteria, but also divide each group into subgroups for analysis. Previous research on relatively large cohorts also showed that plots of female BMI against ART outcome showed an inverted U or J shape. Fedorcsák et al. found that increased BMI in women receiving IVF or ICSI was associated with a lower live birth rate and a higher incidence of early miscarriage (2), thin women had a similar risk of spontaneous abortion, whereas there was progressive increase in risk among overweight, obese, and very obese groups ; normal ; overweight ; and obese . Bellver et al. concluded that the cumulative pregnancy rate after four IVF cycles was reduced as BMI increased. Rittenberg et al. suggest that raised BMI is associated with adverse pregnancy outcome in women undergoing IVF/ICSI treatment, including lower live birth rate which was partially consistent with our study  program, increasing the proportion of normal responders can increase the live birth rate. Assessing ovarian reserve may be helpful in predicting adverse reactions to ovarian hyperstimulation for IVF, and both AFC and serum anti-Müllerian hormone (AMH) levels are indicators that accurately predict ovarian response . MoreoveAccording to our results, the number of fresh embryos transferred was an important clinical factor influencing the live birth rate; that associated with the transfer of two fresh embryos was higher than that associated with the transfer of one embryo. A meta-analysis indicated that double-embryo transfer presented a significantly higher live birth rate than single-embryo transfer . In receIn practical ART clinic work, how to choose a suitable ovarian stimulation protocol for patients with endometriosis is still controversial. In 2010, Sallam et al. published a study in the Cochrane Database, which combined the results of three prospective clinical studies and concluded that 3–6 months of pre-treatment with a GnRH agonist could increase the IVF pregnancy rate of patients with endometriosis by 4-fold and increase the IVF live birth rate of patients with endometriosis by 9-fold . In 2020Our study had some limitations. First, it was a retrospective study, and medical records might be limited and selection bias existed. Second, the sample size was relatively small and could not exclude all potential biases. Our conclusions need to be tested further in prospective, large-scale multicenter clinical trials.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the School of Medicine, Shandong University (No. 2019-1-14). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.JM and LY contributed to conception and design of the study. WL, TS, and YH organized the database. WL and ZG performed the statistical analysis. WL wrote the first draft of the manuscript. LY, ZG, TS, and YH wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: According to recommendations, non-invasive monitoring during neonatal resuscitation after birth includes heart rate (HR) and oxygen saturation (SpO2). Continuous transcutaneous monitoring of carbon dioxide partial pressure (tcpCO2) may further offer quantitative information on neonatal respiratory status.Objective: We aimed to investigate feasibility of tcpCO2 measurements in the delivery room during immediate neonatal transition and to compare the course of tcpCO2 between stable term and preterm infants.Methods: Neonates without need for cardio-respiratory intervention during immediate transition after birth were enrolled in a prospective observational study. In these term and preterm neonates, we measured HR and SpO2 by pulse oximetry on the right wrist and tcpCO2 with the sensor applied on the left hemithorax during the first 15 min after birth. Courses of tcpCO2 were analyzed in term and preterm neonates and groups were compared.Results: Fifty-three term  and 13 preterm neonates  were included. First tcpCO2 values were achieved in both groups at minute 4 after birth, which reached a stable plateau after the equilibration phase at minute 9. Mean tcpCO2 values 15 min after birth were 46.2 (95% CI 34.5–57.8) mmHg in term neonates and 48.5 (95%CI 43.0–54.1) mmHg in preterm neonates. Preterm and term infants did not show significant differences in the tcpCO2 values at any time point.Conclusion: This study demonstrates that tcpCO2 measurement is feasible during immediate neonatal transition after birth and that tcpCO2 values were comparable in stable term and preterm neonates. Neonatologist are confronted with the complexity of monitoring transition from intra- to extra-uterine life. It is a multi-step event, characterized by rapid physiological changes in respiratory, cardio-circulatory and metabolic status.2) followed by respiratory acidosis is one of the main triggers for initiation of breathing . It is recommended as standard practice in the delivery room (DR) by current international guidelines  and peripheral oxygen saturation SpO. It is ridelines , 4.2 and continuous pCO2 monitoring in the DR. Moreover, uncertainty remains about its feasibility and reliability in this setting.Although gas exchange problems may be frequent events during immediate post-natal transition, there is currently no evidence on the role of CO2 (tcpCO2) monitoring in stable neonates during the first 15 min after birth. Moreover, we compared tcpCO2 values between term and preterm neonates without need for any intervention immediately after birth. We hypothesized that tcpCO2 monitoring during immediate transition after birth is feasible and that term and preterm neonates differ in tcpCO2 during this phase.In this study we aimed to evaluate the feasibility of transcutaneous pCOA prospective observational study was conducted from October 2015 to June 2018 at the Division of Neonatology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Austria. Institutional ethical approval (EC number: 27-465 ex 14/15) and written parental consent were obtained before inclusion. Term and preterm neonates born by cesarean section without any intervention immediately after birth were included in the study. Exclusion criteria for this analysis were congenital malformations and/or need for cardio-respiratory support.2 were measured using the IntelliVue MP50 monitor . The tcpCO2 sensor  was applied on the infant's left hemithorax and values were displayed on a separate monitor . In accordance with the manufacturer's guidelines, the sensor's temperature was set at 39°C, the lowest level for tcpCO2 measurements. The fixation of the tcpCO2 sensor was observed continuously and data were only recorded when the sensor was in place and remained attached to the skin.Monitoring was performed during the first 15 min after birth. As soon as the neonate was placed on the resuscitation table in supine position, a pulse oximeter probe was applied on the right wrist and HR and SpOt-test or Mann Whitney U-test, as appropriate. tcpCO2, SpO2, and HR data are presented as mean and 95% confidence interval (95% CI). We investigated tcpCO2, SpO2, and HR across time within the first 15 min using a linear mixed model with fixed effects for time and gestational age (preterm vs. term neonates). A first order autoregressive covariance structure was used. The autoregressive covariance structure assumes a systematically decreasing correlation with increasing distance between time points. Therefore, adjacent time points have the highest correlations. Post-hoc analyses for differences between groups at each minute were performed for the comparison of gestational age groups (pre-term vs. term neonates). A p-value < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS Statistics 24.0.0 .Demographic variables are presented as absolute counts, mean and standard deviation (SD) or median and interquartile range (IQR), as appropriate. Comparisons of categorical baseline characteristics between preterm and term neonates were made using chi-square test, Out of 197 measured neonates in the DR, this study included 53 term and 13 preterm neonates without any cardio-respiratory intervention immediately after birth .2 values were analyzed from minute 4 after birth, which we considered the first proper detection time. Earlier values were detected in just a few patients; thus, they were not included in the analysis. Values reached a stable plateau after the equilibration phase at minute 9. Mean tcpCO2 values 15 min after birth were 46.2 (95% CI 34.5–57.8) mmHg in term neonates and 48.5 (43.0–54.1) mmHg in preterm infants. They both showed comparable courses of tcpCO2 without significant differences at any time point (p = 0.812) (Transcutaneous pCO= 0.812) .2 and HR values were within targets  and HR values were significantly lower at minute 2 .SpO2 measurements in term and preterm neonates in the DR during immediate post-natal transition . Two non-invasive methods for pCO2 monitoring are available: the transcutaneous measurement and the exhaled or end-tidal CO2 (etCO2) measurement .IB, GP, and BU: conception and design. IB, MB, CM, NB-S, BS, LM, and GP: collection and assembly of data. IB, MB, CM, NB-S, BS, LM, AA, BU, and GP: analyses and interpretation of data, critical revision, editing, and final approval of the article. IB, AA, and GP: drafting of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antenatal corticosteroids (ACS) treatment is critical to support survival and lung maturation in preterm infants, however, its effect on feeding and growth is unclear. Prior preterm delivery, it remains uncertain whether ACS treatment should be continued if possible (repeated course ACS), until a certain gestational age is reached. We hypothesized that the association of single-course ACS with feeding competence and postnatal growth outcomes might be different from that of repeated course ACS in very-low-birth-weight preterm infants.A multicenter retrospective cohort study was conducted in very-low-birth-weight preterm infants born at 23–37 weeks’ gestation in South China from 2011 to 2014. Data on growth, nutritional and clinical outcomes were collected. Repeated course ACS was defined in this study as two or more courses ACS (more than single-course). Infants were stratified by gestational age (GA), including GA < 28 weeks, 28 weeks ≤ GA < 32 weeks and 32 weeks ≤ GA < 37 weeks. Multiple linear regression and multilevel model were applied to analyze the association of ACS with feeding and growth outcomes.A total of 841 infants were recruited. The results, just in very-low-birth-weight preterm infants born at 28–32 weeks’ gestation, showed both single and repeated course of ACS regimens had shorter intubated ventilation time compared to non-ACS regimen. Single-course ACS promoted the earlier application of amino acid and enteral nutrition, and higher rate of weight increase  than non-ACS after adjusting for potential confounding factors. No associations of repeated course ACS with feeding, mean weight and weight increase rate were observed.Single-course ACS was positively related to feeding and growth outcomes in very-low-birth-weight preterm infants born at 28–32 weeks’ gestation. However, the similar phenomenon was not observed in the repeated course of ACS regimen. Antenatal corticosteroids (ACS) treatment is commonly used worldwide as the standard treatment for pregnant women at risk of preterm labor . EuropeaThe effects of ACS were different between single and repeated course treatments. Meta-analyses , 9 showeIn summary, we aimed to explored whether ACS, with single-course or repeated course were associated with growth, nutritional outcomes  and clinical outcomes  in VLBW infants based on a retrospective cohort.The retrospective cohort study was a part of the NEOMUNE study , which w− 1·d− 1 [− 1·d − 1) = [1000 × ln (Weight Day n/Weight Day 1)] / (Day n – Day 1). All data were collected until 37 postmenstrual age or at discharge , or death.The VLBW preterm infants were classified as non- ACS group, single-course ACS group and repeated course ACS group. Since a systematic review reported that the scope of GA for which the ACS provides benefits has been controversial . In the − 1·d− 1 . IUGR wa− 1·d− 1 . The rec− 1·d− 1 . Growth H test and χ2 test were used to compare the nutritional and clinical outcomes among ACS groups. Mann–Whitney test was used for post hoc analysis. Multiple linear regression was used to analyze the association of ACS with nutritional and clinical outcomes. Multilevel model was used to analyze the association between ACS and early weight growth trajectories. In multilevel model, the coefficients for ACS were related to differences in mean weight, for interactions between the ACS and time could be interpreted to indicate differences in mean weight varied over time (weight increase rate).All statistical analyses were performed using the software SPSS 25.0 . Cases with lost (or missed) ACS information were eliminated. Data were summarized using means and SDs, medians and interquartile ranges (IQRs), numbers and percentages, as appropriate. Kruskal-Wallis Data in this study were collected from routine records of clinical systems, for this reason, the proportions of missing data in each variable were small, including 0.95% for maternal age, 0.59% for multiple births, 0.23% for cesarean section, 3.68% for birth length, 0.95% for APGAR score, 0.83% for intubation ventilation, 1.55% for non-invasive ventilation, 2.85% for extra oxygen supply, 3.92% for regain birthweight, 0.24% for inpatient time, and 0.59% for amino acid introduction and duration. Multiple imputation was not performed.We used stratified analysis to address confounding factor GA, double entry to reduce information bias and missing data bias. Additionally, the inclusion and exclusion criteria were strictly followed to avoid selection bias. The intrauterine status of some unmeasured confounding may have an impact on nutrition and growth outcomes. In order to ascertain the sensitivity of our results to different health status in utero, analyses were performed to evaluate the association of ACS regimens with nutritional outcomes and postnatal growth in IUGR or non-IUGR VLBW preterm infants in A total of 1178 VLBW preterm infants were identified. Two hundred forty-six cases were excluded for major congenital abnormalities, metabolic diseases or transfer to another hospital within 24 h of birth. Ninety-one cases without ACS information were eliminated. As a result, 841 VLBW preterm infants were recruited in the study, including 456 (54.2%) in non-ACS group, 162 (19.3%) in single-course group and 223 (26.5%) in repeated course group. Average follow-up time was 39 days. The median GA and birthweight were 30.1 (28.9–31.6) weeks and 1300 (1150–1400) g, respectively.In terms of nutrition outcomes, compared with non-ACS group, single-course ACS group had higher proportion of complete breastfeeding in feeding pattern in GA < 28 weeks, and had significant earlier application of amino acid and enteral nutrition in 28 weeks ≤ GA < 32 weeks. We also found repeated course ACS group had larger proportion of mixed feeding, and less 100% preterm formula milk in feeding pattern in 28 weeks ≤ GA < 32 weeks (Table Pertaining to clinical outcomes, both single-course ACS group and repeated course ACS group had shorter time on intubation ventilation support compared with non-ACS group. Repeated course group had the highest rate of cesarean section among the three groups only in 28 weeks ≤ GA < 32 weeks. No differences in clinical outcomes were found between single-course ACS group and repeated course ACS group Table .The weight growth trajectories of VLBW preterm infants were shown in Fig. In unadjusted model, single-course ACS group had a greater average weight over time compared with the repeated course ACS group. Also, single-course ACS and repeated course ACS groups demonstrated higher weight increase rate over time compared with the non-ACS group. After adjusting for confounding factors of birth demographics, mode of delivery, nutritional and respiratory-related indicators, single-course ACS group had higher weight increase rate compared with non-ACS group, however, there were no differences in mean weight among groups. Finally, identical growth advantage for single-course ACS were observed in VLBW preterm infants born at 28–32 weeks’ gestation. No differences in mean weight and weight growth rate among non-ACS, single-course and repeated course ACS treatments were found in other GA group in multivariable analysis.Previous studies showed unclear effects of ACS on postnatal growth and feeding in VLBW preterm infants. The present study showed a close relationship between single-course ACS application and the remarkable improvement in terms of amino acid introduction, enteral nutrition introduction, and weight increase rate in VLBW infants born at 28–32 GA. Additionally, both single and repeated course of ACS regimens had shorter intubated ventilation time compared to non-ACS regimen.Single-course ACS positively correlated with earlier application of amino acid and enteral nutrition with VLBW preterm infants with 28–32 GA. On the contrary, repeated course ACS did not show any association in all GA groups. Previous studies showed that ACS improved the bowel maturation in a manner similar to the lung, that is due to its gastrointestinal tract maturation enhancement effect and anti-inflammatory properties , 26. AniThe results from the present study showed that although there was no difference in birthweight among non-ACS, single-course ACS and repeated course ACS groups, postnatal growth rate was found to be different. Single-course ACS group had higher weight increase rate in infants born at 28–32 GA, however, repeated course ACS did not improve the average weight and weight increase rate during hospitalization after adjusting for sex, cesarean section rate, 5 min APGAR score, GA, multiple birth and nutritional factors. Prior animal and human studies have suggested that multiple repeated ACS may impair fetal growth , 30 or hVLBW preterm infants could experience ‘catch-up’ growth after birth over the weeks and months . InteresBoth single-course and repeated course ACS were associated with shorter time of intubation ventilation in infants born at 28–32 GA, which was consistent with previous researches , 36–38. A major strength of this study was that we continuously measured the weight gain of VLBW preterm infants during hospitalization, compared with previous studies focusing on separate time points. Our study focused on nutrition-related outcomes during hospitalization, while it was less frequently discussed in previous studies. We found that single-course ACS treatment had the greatest benefit for VLBW infants born at 28–32 weeks, suggesting that more cautions are needed when expanding the use of ACS in clinical practice.Limitations in the study included that it was a retrospective study in design, with some potentially confounders unmeasured or uncollected, such as specific data of ACS use, maternal history and sociodemographic characteristics. Furthermore, some parents required an early discharge of their infants subsequently some related clinical outcomes were unclear. We did not make a detailed distinction between one repeat course or multiple weekly repeat course in this study, since the questionnaire was designed and the data were collected during 2011–2014, when there were no guideline recommendations for the use of repeated course ACS. The ACS used in these hospitals was dexamethasone, therefore the final conclusions could not represent other medicine treatment regimen.In summary, single-course ACS treatment was positively related to parenteral and enteral feeding, as well as weight growth in VLBW preterm infants born at 28–32 weeks’ gestation. However, no associations of repeated course ACS with feeding and growth were observed, which indicates that more attention are needed to pay when expanding the use of ACS in clinical practice.Additional file 1.


Patients with paraurethral cyst may be asymptomatic or bothered by sensation of a mass, pain, distorted urinary outflow, dyspareunia, and dysuria (1). Differential diagnosis includes ectopic ureterocele, pelvic organ prolapse, and urethral diverticulum. At present, the management of paraurethral cysts is unclear, but surgical excision appears as the best treatment option (1-3). Alternative methods include waiting for spontaneous rupture, needle aspiration and marsupialization (4). The aim of the video-tutorial is to provide anatomic views and surgical steps necessary to achieve a successful complete excision of a paraurethral cyst. A 54-year-old woman with a 2cm paraurethral cyst bothered by intermittent sensation of an introital mass, dyspareunia, and dysuria was admitted to surgical excision according to the described technique. Urethrocystoscopy and ultrasonography were preoperatively performed to confirm the diagnosis and rule out an urethral diverticulum. Surgical steps included: cyst exposure; vaginal mucosa incision; adequate dissection  with scissors and swab, isolation and excision of paraurethral cyst, layered reconstruction with avoidance of suture layers overlapping. Surgical procedure was successfully achieved without complications. The postoperative course was uneventful. No recurrence was observed and the patient reported complete resolution of her symptoms. The featured video showed complete excision of a paraurethral cyst successfully achieved without complications. Surgical excision represents a safe and effective procedure to manage paraurethral cysts. This step-by-step video-tutorial may represent an important tool to improve surgical know-how.