Comparison of luteal phase and follicular phase in-vitro maturation in women with oocyte maturation abnormalities.

RESEARCH QUESTION: Are there differences in immature oocyte retrieval following luteal phase in-vitro maturation (IVM) compared with follicular phase IVM in women with oocyte maturation abnormalities (OMAs). DESIGN: From January 2019 to May 2023, a retrospective cohort study at a private IVF centre included 36 women with 53 IVM cycles in Group 1 (follicular phase) and 24 women with 32 IVM cycles in Group 2 (luteal phase). Additionally, nine women had both follicular and luteal phase IVM cycles for intracycle variability analysis. RESULTS: There were no differences in oocyte maturation stages between the groups at collection. Group 1 and Group 2 exhibited comparable median metaphase II oocyte rates per patient at 48 h after collection [40.0%, interquartile range (IQR) 0.0-66.7% versus 22.5%, IQR 0.0-52.9%] (P = 0.53). The median fertilization rate in Group 1 (66.7%, IQR 50.0-66.7%) was found to be comparable with that in Group 2 (66.7%, IQR 50.0-66.7%). There were no significant differences in the yielded embryo grades and pregnancy rates between the groups. Comparing follicular and luteal phase IVM within the same menstrual cycle in nine patients, no differences were observed in metaphase II oocyte maturation rates (P > 0.05). CONCLUSIONS: This study found no significant differences in oocyte maturation, fertilization rate, embryo quality or pregnancy outcomes between luteal phase and follicular phase IVM in women with OMAs. These findings suggest that luteal phase IVM can be used similarly to follicular phase IVM, offering a potential avenue to enhance embryo yield for women with OMAs.

Oocyte in-vitro maturation primed with letrozole-HCG versus FSH-HCG in women with oocyte maturation abnormalities: a retrospective study.

RESEARCH QUESTION: Are there differences between in-vitro maturation (IVM) primed with letrozole-human chorionic gonadotrophin (HCG) and IVM primed with FSH-HCG in women with oocyte maturation abnormalities (OMAs), defined as at least two failed IVF cycles where immature oocytes were retrieved? DESIGN: This retrospective study was conducted at a private fertility clinic from January 2009 to April 2023. The final analysis included 75 women in Group 1 (IVM primed with FSH-HCG) and 52 women in Group 2 (IVM primed with letrozole-HCG). RESULTS: A significantly higher median number of oocytes was obtained in Group 1 compared with Group 2 {9 [interquartile range (IQR) 1-5] versus 5 (IQR 1-18); P < 0.001}. However, no differences in oocyte maturation stage at collection were found between the groups (P > 0.05). At the end of IVM, Group 1 had 73/666 mature oocytes and Group 2 had 106/322 mature oocytes, and the median metaphase II oocyte rate per patient was higher in Group 2 [33.3% (IQR 66.7-100.0%) versus 0.0% (IQR 0.0-22.2%); P < 0.001]. Moreover, Group 2 demonstrated a higher median fertilization rate [66.7% (IQR 50.0-100.0%) versus 50.0% (IQR 0.0-66.7%); P = 0.027]. Group 2 had a higher proportion of Grade 2 embryos (58.5% versus 6.3%), and Group 1 had a higher proportion of Grade 3 embryos (93.8% vs 24.4%; P < 0.001). Notably, all pregnancies obtained in the study were in Group 2 (5 versus 0; P = 0.042). CONCLUSIONS: IVM primed with letrozole-HCG in women with prior failed IVF cycles due to OMAs may result in mature oocytes, clinical pregnancies and live births. The effectiveness of letrozole priming for the subtypes of OMAs needs further investigation, with studies including greater numbers of cases.

The Vagus Nerve Regulates Immunometabolic Homeostasis in the Ovine Fetus near Term: The Impact on Terminal Ileum.

BACKGROUND: Glucosensing elements are widely distributed throughout the body and relay information about circulating glucose levels to the brain via the vagus nerve. However, while anatomical wiring has been established, little is known about the physiological role of the vagus nerve in glucosensing. The contribution of the vagus nerve to inflammation in the fetus is poorly understood. Increased glucose levels and inflammation act synergistically when causing organ injury, but their interplay remains incompletely understood. We hypothesized that vagotomy (Vx) will trigger a rise in systemic glucose levels and this will be enhanced during systemic and organ-specific inflammation. Efferent vagus nerve stimulation (VNS) should reverse this phenotype. METHODS: Near-term fetal sheep (n = 57) were surgically prepared using vascular catheters and ECG electrodes as the control and treatment groups (lipopolysaccharide (LPS), Vx + LPS, Vx + LPS + selective efferent VNS). The experiment was started 72 h postoperatively to allow for post-surgical recovery. Inflammation was induced with LPS bolus intravenously (LPS group, 400 ng/fetus/day for 2 days; n = 23). For the Vx + LPS group (n = 11), a bilateral cervical vagotomy was performed during surgery; of these n = 5 received double the LPS dose, LPS800. The Vx + LPS + efferent VNS group (n = 8) received cervical VNS probes bilaterally distal from Vx in eight animals. Efferent VNS was administered for 20 min on days 1 and 2 +/10 min around the LPS bolus. Fetal arterial blood samples were drawn on each postoperative day of recovery (-72 h, -48 h, and -24 h) as well as at the baseline and seven selected time points (3-54 h) to profile inflammation (ELISA IL-6, pg/mL), insulin (ELISA), blood gas, and metabolism (glucose). At 54 h post-LPS, a necropsy was performed, and the terminal ileum macrophages' CD11c (M1 phenotype) immunofluorescence was quantified to detect inflammation. The results are reported for p < 0.05 and for Spearman R2 > 0.1. The results are presented as the median (IQR). RESULTS: Across the treatment groups, blood gas and cardiovascular changes indicated mild septicemia. At 3 h in the LPS group, IL-6 peaked. That peak was decreased in the Vx + LPS400 group and doubled in the Vx + LPS800 group. The efferent VNS sped up the reduction in the inflammatory response profile over 54 h. The M1 macrophage activity was increased in the LPS and Vx + LPS800 groups only. The glucose and insulin concentrations in the Vx + LPS group were, respectively, 1.3-fold (throughout the experiment) and 2.3-fold higher vs. control (at 3 h). The efferent VNS normalized the glucose concentrations. CONCLUSIONS: The complete withdrawal of vagal innervation resulted in a 72-h delayed onset of a sustained increase in glucose for at least 54 h and intermittent hyperinsulinemia. Under the conditions of moderate fetal inflammation, this was related to higher levels of gut inflammation. The efferent VNS reduced the systemic inflammatory response as well as restored both the concentrations of glucose and the degree of terminal ileum inflammation, but not the insulin concentrations. Supporting our hypothesis, these findings revealed a novel regulatory, hormetic, role of the vagus nerve in the immunometabolic response to endotoxin in near-term fetuses.

Factors Determining the Success of the Chronically Instrumented Non-anesthetized Fetal Sheep Model of Human Development: A Retrospective Cohort Study.

Background The chronically instrumented non-anesthetized fetal sheep (CINAFS) model has been a mainstay of human fetal development research for the past 60 years. As a large "two for one" animal model, involving the instrumentation of the ewe and her fetus, the model poses challenges to implement de novo and maintain overtime at the highest standards of operating procedures to ensure ongoing performance. A common yet conventionally underreported issue researchers face is a high rate of animal loss. Here, we investigate what determines the success of the CINAFS model of human development. Methods We conducted a retrospective cohort analysis consisting of 82 experiments spanning the course of six years. Our team identified 10 variables that we anticipated were likely to influence the experimental outcome, such as the time of year, animal size, and surgical complexity. To evaluate the role of each variable in contributing to the success of the model, a binary logit regression analysis with a Fisher scoring optimization was fit to the data (SAS, V9 engine, release 3.8, SAS Institute, Cary, NC, USA). A higher predictive probability indicates a larger impact by the given variable on the outcome of the experiment. A Wald chi-squared analysis was run on the data to control for confounders and determine significance. Results The single variable identified in this study as determining the success of experiment outcomes using the CINAFS model is the experience level of the team. Conclusion The CINAFS model offers enormous potential to further our understanding of human fetal development and create interventional technologies related to fetal health. However, to improve experimental outcomes using the CINAFS model, stronger communication and training are needed. We discuss the implications of our findings for the successful implementation of this challenging yet scientifically advantageous animal model of human physiology.

Recording and manipulation of vagus nerve electrical activity in chronically instrumented unanesthetized near term fetal sheep.

BACKGROUND: The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli. This is due to the unique amenability of the unanesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation, and in vivo organ imaging. Recently, there has been growing interest in the pleiotropic effects of vagus nerve stimulation (VNS) on various organ systems such as innate immunity and inflammation, and metabolism. There is no approach to study this in utero and corresponding physiological understanding is scarce. NEW METHOD: Based on our previous presentation of a stable chronically instrumented unanesthetized fetal sheep model, here we describe the surgical instrumentation procedure allowing successful implantation of a cervical uni- or bilateral VNS probe with or without vagotomy. RESULTS: In a cohort of 68 animals, we present the changes in blood gas, metabolic, and inflammatory markers during the postoperative period. We detail the design of a VNS probe which also allows recording from the fetal nerve. We also present an example of fetal vagus electroneurogram (VENG) recorded from the VNS probe and an analytical approach to the data. COMPARISON WITH EXISTING METHODS: This method represents the first implementation of fetal VENG/VNS in a large pregnant mammalian organism. CONCLUSIONS: This study describes a new surgical procedure allowing to record and manipulate chronically fetal vagus nerve activity in an animal model of human pregnancy.

High-resolution analyses of human sperm dynamic methylome reveal thousands of novel age-related epigenetic alterations.

BACKGROUND: Children of aged fathers are at a higher risk of developing mental disorders. Alterations in sperm DNA methylation have been implicated as a potential cause. However, age-dependent modifications of the germ cells' epigenome remain poorly understood. Our objective was to assess the DNA methylation profile of human spermatozoa during aging. RESULTS: We used a high throughput, customized methylC-capture sequencing (MCC-seq) approach to characterize the dynamic DNA methylation in spermatozoa from 94 fertile and infertile men, who were categorized as young, 48 men between 18-38 years or old 46 men between 46-71 years. We identified more than 150,000 age-related CpG sites that are significantly differentially methylated among 2.65 million CpG sites covered. We conducted machine learning using our dataset to predict the methylation age of subjects; the age prediction accuracy based on our assay provided a more accurate prediction than that using the 450 K chip approach. In addition, we found that there are more hypermethylated (62%) than hypomethylated (38%) CpG sites in sperm of aged men, corresponding to 798 of total differential methylated regions (DMRs), of which 483 are hypermethylated regions (HyperDMR), and 315 hypomethylated regions (HypoDMR). Moreover, the distribution of age-related hyper- and hypomethylated CpGs in sperm is not random; the CpG sites that were hypermethylated with advanced age were frequently located in the distal region to genes, whereas hypomethylated sites were near to gene transcription start sites (TSS). We identified a high density of age-associated CpG changes in chromosomes 4 and 16, particularly HyperDMRs with localized clusters, the chr4 DMR cluster overlaps PGC1α locus, a protein involved in metabolic aging and the chr16 DMR cluster overlaps RBFOX1 locus, a gene implicated in neurodevelopmental disease. Gene ontology analysis revealed that the most affected genes by age were associated with development, neuron projection, differentiation and recognition, and behaviour, suggesting a potential link to the higher risk of neurodevelopmental disorders in children of aged fathers. CONCLUSION: We identified thousands of age-related and sperm-specific epigenetic alterations. These findings provide novel insight in understanding human sperm DNA methylation dynamics during paternal aging, and the subsequently affected genes potentially related to diseases in offspring.

Vagal contributions to fetal heart rate variability: an omics approach.

OBJECTIVE: Fetal heart rate variability (fHRV) is an important indicator of health and disease, yet its physiological origins, neural contributions, in particular, are not well understood. We aimed to develop novel experimental and data analytical approaches to identify fHRV measures reflecting the vagus nerve contributions to fHRV. APPROACH: In near-term ovine fetuses, a comprehensive set of 46 fHRV measures was computed from fetal pre-cordial electrocardiogram recorded during surgery and 72 h later without (n = 24) and with intra-surgical bilateral cervical vagotomy (n = 15). MAIN RESULTS: The fetal heart rate did not change due to vagotomy. We identify fHRV measures specific to the vagal modulation of fHRV: multiscale time irreversibility asymmetry index (AsymI), detrended fluctuation analysis (DFA) α 1, Kullback-Leibler permutation entropy (KLPE) and scale-dependent Lyapunov exponent slope (SDLE α). SIGNIFICANCE: We provide a systematic delineation of vagal contributions to fHRV across signal-analytical domains which should be relevant for the emerging field of bioelectronic medicine and the deciphering of the 'vagus code'. Our findings also have clinical significance for in utero monitoring of fetal health during surgery. Key points •Fetal surgery causes a complex pattern of changes in heart rate variability measures with an overall reduction of complexity or variability. •At 72 h after surgery, many of the HRV measures recover and this recovery is delayed by an intrasurgical cervical bilateral vagotomy. •We identify HRV pattern representing complete vagal withdrawal that can be understood as part of 'HRV code', rather than any single HRV measure. •We identify HRV biomarkers of recovery from fetal surgery and discuss the effect of anticholinergic medication on this recovery.

α7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin.

Neuroinflammation in utero may result in lifelong neurological disabilities. Astrocytes play a pivotal role in this process, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of astrocytes. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal astrocytes will augment their neuroprotective transcriptome profile, while the inhibition of α7nAChR will achieve the opposite. Using an in vivo-in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep astrocytes cultures re-exposed to LPS in the presence of a selective α7nAChR agonist or antagonist. Our RNAseq findings show that a pro-inflammatory astrocyte transcriptome phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, α7nAChR inhibition potentiates the pro-inflammatory astrocytic transcriptome phenotype. Furthermore, we conducted a secondary transcriptome analysis against the identical α7nAChR experiments in fetal sheep primary microglia cultures. Similar to findings in fetal microglia, in fetal astrocytes we observed a memory effect of in vivo exposure to inflammation, expressed in a perturbation of the iron homeostasis signaling pathway (hemoxygenase 1, HMOX1), which persisted under pre-treatment with α7nAChR antagonist but was reversed with α7nAChR agonist. For both glia cell types, common pathways activated due to LPS included neuroinflammation signaling and NF-κB signaling in some, but not all comparisons. However, overall, the overlap on the level of signaling pathways was rather minimal. Astrocytes, not microglia-the primary immune cells of the brain, were characterized by unique inhibition patterns of STAT3 pathway due to agonistic stimulation of α7nAChR prior to LPS exposure. Lastly, we discuss the implications of our findings for fetal and postnatal brain development.

Sculpting the Sculptors: Methods for Studying the Fetal Cholinergic Signaling on Systems and Cellular Scales.

The non-neuronal, immunological effects of the cholinergic signaling are exerted on the system's scale of observation via the vagus nerve and on the cellular scale via α7 nicotinic acetylcholine receptor (nAChR) signaling in myeloid cells of the periphery or brain's microglia and astrocytes. The developmental effects of such multi-scale signaling can be conceived of as an example of psychoneuroimmunological (PNI) homeokinesis and, while reported in the literature, are not yet systematically well studied. To be better understood, the intricacy of the multi-scale interactions requires relevant preclinical animal models. Chronically instrumented non-anesthetized fetal sheep model comes with a strong track record of bench-to-bed translation and a large body of evidence for its strong resemblance to and relevance for human physiology on various scales of organization. Recently, there has been growing interest in pleiotropic effects of vagus nerve stimulation (VNS) on various organ systems such as innate immunity, metabolism, and emotion with implications for programming of PNI phenotype. Here we describe the procedures required to record and manipulate the vagus nerve activity in this large pregnant mammalian organism. Extending this in vivo model to in vitro, on the cellular scale, we present the method to manipulate the cholinergic signaling in ovine fetal microglia and astrocytes and analyze their responses on protein and RNA levels. Together these models can provide multi-scale-level mechanistic insights into the effects of cholinergic signaling on PNI phenotype.

Temporal Patterns in Sheep Fetal Heart Rate Variability Correlate to Systemic Cytokine Inflammatory Response: A Methodological Exploration of Monitoring Potential Using Complex Signals Bioinformatics.

Fetal inflammation is associated with increased risk for postnatal organ injuries. No means of early detection exist. We hypothesized that systemic fetal inflammation leads to distinct alterations of fetal heart rate variability (fHRV). We tested this hypothesis deploying a novel series of approaches from complex signals bioinformatics. In chronically instrumented near-term fetal sheep, we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 10) observing it over 54 hours; seven additional fetuses served as controls. Fifty-one fHRV measures were determined continuously every 5 minutes using Continuous Individualized Multi-organ Variability Analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. We implemented, validated and tested methodology to obtain a subset of CIMVA fHRV measures that matched best the temporal profile of the inflammatory cytokine IL-6. In the LPS group, IL-6 peaked at 3 hours. For the LPS, but not control group, a sharp increase in standardized difference in variability with respect to baseline levels was observed between 3 h and 6 h abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. We derived fHRV inflammatory index (FII) consisting of 15 fHRV measures reflecting the fetal inflammatory response with prediction accuracy of 90%. Hierarchical clustering validated the selection of 14 out of 15 fHRV measures comprising FII. We developed methodology to identify a distinctive subset of fHRV measures that tracks inflammation over time. The broader potential of this bioinformatics approach is discussed to detect physiological responses encoded in HRV measures.

Can Monitoring Fetal Intestinal Inflammation Using Heart Rate Variability Analysis Signal Incipient Necrotizing Enterocolitis of the Neonate?

OBJECTIVE: Necrotizing enterocolitis of the neonate is an acute inflammatory intestinal disease that can cause necrosis and sepsis. Chorioamnionitis is a risk factor of necrotizing enterocolitis. The gut represents the biggest vagus-innervated organ. Vagal activity can be measured via fetal heart rate variability. We hypothesized that fetal heart rate variability can detect fetuses with incipient gut inflammation. DESIGN: Prospective animal study. SETTING: University research laboratory. SUBJECTS: Chronically instrumented near-term fetal sheep (n = 21). MEASUREMENTS AND MAIN RESULTS: Animals were surgically instrumented with vascular catheters and electrocardiogram to allow manipulation and recording from nonanesthetized animals. In 14 fetal sheep, inflammation was induced with lipopolysaccharide (IV) to mimic chorioamnionitis. Fetal arterial blood samples were drawn at selected time points over 54 hours post lipopolysaccharide for blood gas and cytokines (interleukin-6 and tumor necrosis factor-α enzymelinked immunosorbent assay). Fetal heart rateV was quantified throughout the experiment. The time-matched fetal heart rate variability measures were correlated to the levels of interleukin-6 and tumor necrosis factor-α. Upon necropsy, ionized calcium binding adaptor molecule 1+ (Iba1+), CD11c+ (M1), CD206+ (M2 macrophages), and occludin (leakiness marker) immunofluorescence in the terminal ileum was quantified along with regional Iba1+ signal in the brain (microglia). Interleukin-6 peaked at 3 hours post lipopolysaccharide accompanied by mild cardiovascular signs of sepsis. At 54 hours, we identified an increase in Iba1+ and, specifically, M1 macrophages in the ileum accompanied by increased leakiness, with no change in Iba1 signal in the brain. Preceding this change on tissue level, at 24 hours, a subset of nine fetal heart rate variability measures correlated exclusively to the Iba+ markers of ileal, but not brain, inflammation. An additional fetal heart rate variability measure, mean of the differences of R-R intervals, correlated uniquely to M1 ileum macrophages increasing due to lipopolysaccharide. CONCLUSIONS: We identified a unique subset of fetal heart rate variability measures reflecting 1.5 days ahead of time the levels of macrophage activation and increased leakiness in terminal ileum. We propose that such subset of fetal heart rate variability measures reflects brain-gut communication via the vagus nerve. Detecting such noninvasively obtainable organ-specific fetal heart rate variability signature of inflammation would alarm neonatologists about neonates at risk of developing necrotizing enterocolitis and sepsis. Clinical validation studies are required.

Vagus Nerve Stimulation for Treatment of Inflammation: Systematic Review of Animal Models and Clinical Studies.

Vagus nerve stimulation (VNS) has been used since 1997 for treatment of drug-resistant epilepsy. More recently, an off-label use of VNS has been explored in animal models and clinical trials for treatment of a number of conditions involving the innate immune system. The underlying premise has been the notion of the cholinergic antiinflammatory pathway (CAP), mediated by the vagus nerves. While the macroanatomic substrate - the vagus nerve - is understood, the physiology of the pleiotropic VNS effects and the "language" of the vagus nerve, mediated brain-body communication, remain an enigma. Tackling this kind of enigma is precisely the challenge for and promise of bioelectronic medicine. We review the state of the art of this emerging field as it pertains to developing strategies for use of the endogenous CAP to treat inflammation and infection in various animal models and human clinical trials. This is a systematic PubMed review for the MeSH terms "vagus nerve stimulation AND inflammation." We report the diverse profile of currently used VNS antiinflammatory strategies in animal studies and human clinical trials. This review provides a foundation and calls for devising systematic and comparable VNS strategies in animal and human studies for treatment of inflammation. We discuss species-specific differences in the molecular genetics of cholinergic signaling as a framework to understand the divergence in VNS effects between species. Brain-mapping initiatives are needed to decode vagus-carried brain-body communication before hypothesis-driven treatment approaches can be devised.

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

Effect of maternal ketoacidosis on the ovine fetus.

Ketoacidosis during pregnancy carries significant risk of intrauterine fetal demise, but little is known about the impact of ketoacids on the ovine fetus. We report a case series of maternal ketoacidosis in ewes. Maternal ketoacidosis may result in biochemical and acid-base fetal abnormalities associated with changes in feto-placental unit perfusion.

Effet de l'acidocétose maternelle sur un fœtus ovin. L'acidocétose durant la gestation comporte un risque important de mortalité intra-utérine du fœtus, mais on connaît peu de choses à propos de l'impact des acides cétoniques sur le fœtus ovin. Nous signalons une série de cas d'acidocétose maternelle chez les brebis. L'acidocétose maternelle peut provoquer des anomalies biochimiques et acides avec des changements dans la perfusion de l'unité fœto-placentaire.(Traduit par Isabelle Vallières).

Does heart rate variability reflect the systemic inflammatory response in a fetal sheep model of lipopolysaccharide-induced sepsis?

Fetal inflammatory response occurs during chorioamnionitis, a frequent and often subclinical inflammation associated with increased risk for brain injury and life-lasting neurologic deficits. No means of early detection exist. We hypothesized that systemic fetal inflammation without septic shock will be reflected in alterations of fetal heart rate (FHR) variability (fHRV) distinguishing baseline versus inflammatory response states. In chronically instrumented near-term fetal sheep (n = 24), we induced an inflammatory response with lipopolysaccharide (LPS) injected intravenously (n = 14). Ten additional fetuses served as controls. We measured fetal plasma inflammatory cytokine IL-6 at baseline, 1, 3, 6, 24 and 48 h. 44 fHRV measures were determined continuously every 5 min using continuous individualized multi-organ variability analysis (CIMVA). CIMVA creates an fHRV measures matrix across five signal-analytical domains, thus describing complementary properties of fHRV. Using principal component analysis (PCA), a widely used technique for dimensionality reduction, we derived and quantitatively compared the CIMVA fHRV PCA signatures of inflammatory response in LPS and control groups. In the LPS group, IL-6 peaked at 3 h. In parallel, PCA-derived fHRV composite measures revealed a significant difference between LPS and control group at different time points. For the LPS group, a sharp increase compared to baseline levels was observed between 3 h and 6 h, and then abating to baseline levels, thus tracking closely the IL-6 inflammatory profile. This pattern was not observed in the control group. We also show that a preselection of fHRV measures prior to the PCA can potentially increase the difference between LPS and control groups, as early as 1 h post LPS injection. We propose a fHRV composite measure that correlates well with levels of inflammation and tracks well its temporal profile. Our results highlight the potential role of HRV to study and monitor the inflammatory response non-invasively over time.

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation.

OBJECTIVE: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. METHODS: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation. RESULTS: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1ß response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia. DISCUSSION: We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

Adaptive shut-down of EEG activity predicts critical acidemia in the near-term ovine fetus.

In fetal sheep, the electrocorticogram (ECOG) recorded directly from the cortex during repetitive heart rate (FHR) decelerations induced by umbilical cord occlusions (UCO) predictably correlates with worsening hypoxic-acidemia. In human fetal monitoring during labor, the equivalent electroencephalogram (EEG) can be recorded noninvasively from the scalp. We tested the hypothesis that combined fetal EEG - FHR monitoring allows for early detection of worsening hypoxic-acidemia similar to that shown for ECOG-FHR monitoring. Near-term fetal sheep (n = 9) were chronically instrumented with arterial and venous catheters, ECG, ECOG, and EEG electrodes and umbilical cord occluder, followed by 4 days of recovery. Repetitive UCOs of 1 min duration and increasing strength (with regard to the degree of reduction in umbilical blood flow) were induced each 2.5 min until pH dropped to <7.00. Repetitive UCOs led to marked acidosis (arterial pH 7.35 ± 0.01 to 7.00 ± 0.03). At pH of 7.22 ± 0.03 (range 7.32-7.07), and 45 ± 9 min (range 1 h 33 min-20 min) prior to attaining pH < 7.00, both ECOG and EEG amplitudes began to decrease ~fourfold during each FHR deceleration in a synchronized manner. Confirming our hypothesis, these findings support fetal EEG as a useful adjunct to FHR monitoring during human labor for early detection of incipient fetal acidemia.

The Ovine Fetal and Placental Inflammatory Response to Umbilical Cord Occlusions With Worsening Acidosis.

We hypothesized that repetitive umbilical cord occlusions (UCOs) leading to severe acidemia will stimulate a placental and thereby fetal inflammatory response which will be exacerbated by chronic hypoxemia and low-grade bacterial infection. Chronically instrumented fetal sheep served as controls or underwent repetitive UCOs for up to 4 hours or until fetal arterial pH was <7.00. Normoxic-UCO and hypoxic-UCO fetuses had arterial O2 saturation pre-UCOs of >55% and <55%, respectively, while lipopolysaccharide (LPS)-UCO fetuses received LPS intra-amniotic (2 mg/h) starting 1 hour pre-UCOs. Fetal plasma and amniotic fluid were sampled for interleukin (IL) 6 and IL-1ß. Animals were euthanized at 48 hours of recovery with placental cotyledons processed for measurement of macrophage, neutrophil, and mast cell counts. Repetitive UCOs resulted in severe fetal acidemia with pH approaching 7.00 for all 3 UCO groups. Neutrophils, while unchanged within the cotyledon fetal and intermediate zones, were ∼2-fold higher within the zona intima for all 3 UCO groups. However, no differences were observed in macrophage counts among the treatment groups and no cotyledon mast cells were seen. Fetal plasma and amniotic fluid cytokines remained little changed post-UCOs and/or at 1 and 48 hours of recovery in the normoxic-UCO and hypoxic-UCO groups but increased several fold in the LPS-UCO group with IL-6 plasma values at 1 hour recovery highly correlated with the nadir pH attained (r = -.97). As such, repetitive UCOs with severe acidemia can induce a placental inflammatory response and more so with simulated low-grade infection and likely contributing to cytokine release in the umbilical circulation.

Cell fate regulation by gelsolin in human gynecologic cancers.

Chemoresistance is a major hurdle in cancer treatment. Down-regulation of apoptosis pathways is one of the key determinants for chemoresistance. Here, we report higher gelsolin (GSN) levels in chemoresistant gynecological cancer cells compared with their sensitive counterparts. cis-Diammine dichloroplatinium (II) (CDDP)-induced GSN down-regulation is associated with its cleavage and apoptosis. Although the C-terminal GSN fragment (C-GSN) sensitized chemoresistant cells to CDDP, intact GSN and its N-terminal fragment (N-GSN) attenuated this response. GSN silencing also facilitated CDDP-induced apoptosis in chemoresistant cells. In contrast, intact GSN (I-GSN) was prosurvival in the presence of CDDP through a FLICE-like inhibitory protein (FLIP)-Itch interaction. This interaction was colocalized in the perinuclear region that could be dissociated by CDDP in sensitive cells, thereby inducing FLIP ubiquitination and degradation, followed by apoptosis. In resistant cells, GSN was highly expressed and CDDP failed to abolish the I-GSN-FLIP-Itch interaction, resulting in the dysregulation of the downstream responses. In addition, we investigated the association between GSN expression in ovarian serous adenocarcinoma and progression free survival and overall survival, as well as clinical prognosis. GSN overexpression was significantly associated with more aggressive behavior and more cancer deaths and supported our hypothesis that high GSN expression confers chemoresistance in cancer cells by altering the GSN-FLIP-Itch interaction. These findings are in agreement with the notion that GSN plays an important role in the regulation of gynecological cell fate as reflected in dysregulation in chemosensitivity.

Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5) and later (E15.5-17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in the formation of the mural cell layer and weakening of vessel integrity. These results demonstrate that overexpression of progranulin in endothelial cells influences normal angiogenesis in vivo.