Martin G. Frasch

Fetal body weight and the development of the control of the cardiovascular system in fetal sheep

Reduced birth weight predisposes to cardiovascular diseases in later life. We examined in fetal sheep at 0.76 (n= 18) and 0.87 (n= 17) gestation whether spontaneously occurring variations in fetal weight affect maturation of autonomic control of cardiovascular function. Fetal weights at both gestational ages were grouped statistically in low (LW) and normal weights (NW) (P < 0.01). LW fetuses were within the normal weight span showing minor growth dysproportionality at 0.76 gestation favouring heart and brain, with a primary growth of carcass between 0.76 and 0.87 gestation (P < 0.05). While twins largely contributed to LW fetuses, weight differences between singletons and twins were absent at 0.76 and modest at 0.87 gestation, underscoring the fact that twins belong to normality in fetal sheep not constituting a major malnutritive condition. Mean fetal blood pressure (FBP) of all fetuses was negatively correlated to fetal weight at 0.76 but not 0.87 gestation (P < 0.05). At this age, FBP and baroreceptor reflex sensitivity were increased in LW fetuses (P < 0.05), suggesting increased sympathetic activity and immaturity of circulatory control. Development of vagal modulation of fetal heart rate depended on fetal weight (P < 0.01). These functional associations were largely independent of twin pregnancies. We conclude, low fetal weight within the normal weight span is accompanied by a different trajectory of development of sympathetic blood pressure and vagal heart rate control. This may contribute to the development of elevated blood pressure in later life. Examination of the underlying mechanisms and consequences may contribute to the understanding of programming of cardiovascular diseases.

Systemic and cerebral inflammatory response to umbilical cord occlusions with worsening acidosis in the ovine fetus.

OBJECTIVE We hypothesized that repetitive umbilical cord occlusions (UCOs) with worsening acidosis will lead to a fetal inflammatory response. STUDY DESIGN Chronically instrumented fetal sheep underwent a series of UCOs until fetal arterial pH decreased to <7.00. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1B and IL-6 levels. Animals were euthanized at 24 hours of recovery with brain tissue processed for subsequent measurement of microglia and mast cell counts. RESULTS Repetitive UCOs resulted in a severe degree of fetal acidemia. Fetal plasma IL-1B values were increased approximately 2-fold when measured at maximal fetal acidosis and again at 1-2 hours of recovery. Fetal microglia cells were increased approximately 2-fold in the white matter and hippocampus, while mast cells were increased approximately 2-fold in the choroid plexus and now evident in the thalamus when analyzed at 24 hours recovery. CONCLUSION Repetitive UCOs leading to severe acidemia in the ovine fetus near term will result in an inflammatory response both systemically and locally within the brain.

Measures of acidosis with repetitive umbilical cord occlusions leading to fetal asphyxia in the near-term ovine fetus

OBJECTIVE Increasing frequency of severe variable decelerations is associated with acidemia. However, there is little understanding of its development or normalization rate. STUDY DESIGN To quantify the time course of acidemia, 10 near-term fetal sheep underwent a series of mild (1 minute every 5 minutes), moderate (1 minute every 3 minutes), and severe (1 minute every 2 minutes) umbilical cord occlusions (UCO), lasting 1 hour each or until fetal arterial pH decreased to less than 7.00. RESULTS Each minute of UCO resulted in base deficit (BD) and lactate increases at rates of 0.56 and 0.35 mmol/L per minute, respectively. During a 2 h recovery, BD and lactate normalized at rates of 0.09 and 0.04 mmol/L per minute. BD correlated highly with lactate (r = 0.95; P < .001). CONCLUSION Our findings in the near-term ovine fetus suggest that the knowledge of fetal BD deterioration and recovery rates can aid assessing fetal acidemia during labor.

Nonlinear properties of vagal and sympathetic modulations of heart rate variability in ovine fetus near term.

Fetal heart rate (FHR) monitoring is commonly used although clinical studies questioned its diagnostic value. Sophisticated FHR variability (fHRV) measures such as fHRV complexity may improve the sensitivity and specificity of FHR monitoring. A more detailed understanding of the physiology underlying fHRV complexity is essential to harness its use for monitoring fetal health. To examine the specific effects of vagal and sympathetic modulations on fHRV complexity, we blocked vagal activity with atropine and sympathetic activity with propranolol in near-term fetal sheep (n = 7, 0.85 gestation). Under these conditions, we analyzed the linear and nonlinear parts of fHRV complexity from autonomic information flow. Overall fHRV complexity decreased with both drugs compared with nonrapid eye movement sleep baseline (P < 0.05). With atropine, this was because of a decrease of the linear part of fHRV complexity on the long-term time scale (P < 0.05), suggesting that vagal modulation of fHRV is adequately described by linear fHRV measures. With propranolol, the nonlinear part of fHRV complexity decreased on the short-term time scale (P < 0.05), suggesting that sympathetic influences on fHRV can be detected by the nonlinear part of fHRV complexity. Thus the complex interplay of vagal and sympathetic modulations of fHRV is reflected differently and specifically in the linear and nonlinear properties of fHRV complexity, and on different time scales. Analysis of linear and nonlinear properties of fHRV may improve sensitivity and specificity of FHR monitoring.

Sampling rate of heart rate variability impacts the ability to detect acidemia in ovine fetuses near-term.

Background: To evaluate the impact of sampling rate on the predictive capability of continuous fetal heart rate (FHR) variability (fHRV) monitoring for detecting fetal acidemia during labor, we tested the performance of the root mean square of successive differences (RMSSD) in R–R intervals from the ECG when acquired with the sampling rate of 4 Hz currently available in FHR monitors, in comparison to the gold standard of 1000 Hz. Methods: Near-term ovine fetuses (N = 9) were chronically prepared with precordial electrodes for recording ECG, vascular catheters for blood sampling, and an umbilical cord occluder. For 1 min every 2.5 min, animals underwent mild partial umbilical cord occlusions (UCO) × 1 h, moderate partial UCO × 1 h, then complete UCO × 2 h, or until arterial pH reached <7.00. Arterial blood samples were drawn at baseline and every 20 min during the UCO series. RMSSD was calculated continuously in 5 min windows using an automated, standardized system (CIMVA.com). Results are presented as mean ± SEM with significance assumed for p < 0.05. Results: Repetitive UCO resulted in pH decreasing from 7.35 ± 0.01 to 7.00 ± 0.03. In all nine animals, RMSSD increased from 16.7 ± 1.0 ms at baseline to 44.4 ± 2.3 ms, 70 ± 15 min prior to reaching the pH nadir when sampled at 1000 Hz. When sampled at 4 Hz, RMSSD at baseline measured 36.1 ± 6.0 ms and showed no significant increase during the UCO series until the pH nadir was reached. Consequently, early detection of severe hypoxic-acidemia would have been missed in all fetuses. Conclusion: RMSSD as a measure of fHRV when calculated from FHR sampled at 1000 Hz allowed for the early detection of worsening hypoxic-acidemia in each fetus. However, when calculated at the low sampling rate of 4 Hz used clinically, RMSSD remained unchanged until terminally when the nadir pH was reached. For early detection of fetal acidemia during labor, more sensitive means of acquiring FHR are therefore recommended than currently deployed, e.g., trans-abdominal fetal ECG.

Heart rate variability analysis allows early asphyxia detection in ovine fetus.

Fetal heart rate (FHR) monitoring is commonly used to predict asphyxia but clinical and experimental studies have questioned its diagnostic value. We examined the usefulness of fetal heart rate variability (fHRV) measures in detecting early asphyxia using chronically instrumented fetal sheep under normoxic (n = 6) and asphyxic conditions (3 umbilical cord occlusions, n = 6). The occlusions consistently led to pH decreases from 7.35 + 0.01 to 7.09 + 0.03 ( P < .05). FHR showed biphasic deceleration during each occlusion, associated with increasing arterial blood pressure ( P < .05). RMSSD, an index of vagal modulation of fHRV, increased consistently during repeated occlusion induced FHR decelerations ( P < .05). Under normoxic conditions, RMSSD did not change during FHR decelerations and decreased during FHR accelerations ( P < .05). Our results suggest that an increase of RMSSD in association with FHR decelerations reflects initial vagal activation during fetal asphyxia. RMSSD may accurately identify asphyxic fetuses early. Clinical validation is needed.

Correlation of arterial fetal base deficit and lactate changes with severity of variable heart rate decelerations in the near-term ovine fetus

OBJECTIVE Recent guidelines classify variable decelerations without detail as to degree of depth. We hypothesized that variable deceleration severity is highly correlated with fetal base deficit accumulation. STUDY DESIGN Seven near-term fetal sheep underwent a series of graded umbilical cord occlusions resulting in mild (30 bpm decrease), moderate (60 bpm decrease), or severe (decrease of 90 bpm to baseline <70 bpm) variable decelerations at 2.5 minute intervals. RESULTS Mild, moderate, and severe variable decelerations increased fetal base deficit (0.21 ± 0.03, 0.27 ± 0.03, and 0.54 ± 0.09 mEq/L per minute) in direct proportion to severity. During recovery, fetal base deficit cleared at 0.12 mEq/L per minute. CONCLUSION In this model, ovine fetuses can tolerate repetitive mild and moderate variable decelerations with minimal change in base deficit and lactate. In contrast, repetitive severe variable decelerations may result in significant base deficit increases, dependent on frequency. Modified guideline differentiation of mild/moderate vs severe variable decelerations may aid in the interpretation of fetal heart rate tracings and optimization of clinical management paradigms.

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-1β 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 HMOX1down and FBPup 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.

Acceleration and Deceleration Capacity of Fetal Heart Rate in an In-Vivo Sheep Model

Background Fetal heart rate (FHR) variability is an indirect index of fetal autonomic nervous system (ANS) integrity. FHR variability analysis in labor fails to detect early hypoxia and acidemia. Phase-rectified signal averaging (PRSA) is a new method of complex biological signals analysis that is more resistant to non-stationarities, signal loss and artifacts. It quantifies the average cardiac acceleration and deceleration (AC/DC) capacity. Objective The aims of the study were: (1) to investigate AC/DC in ovine fetuses exposed to acute hypoxic-acidemic insult; (2) to explore the relation between AC/DC and acid-base balance; and (3) to evaluate the influence of FHR decelerations and specific PRSA parameters on AC/DC computation. Methods Repetitive umbilical cord occlusions (UCOs) were applied in 9 pregnant near-term sheep to obtain three phases of MILD, MODERATE, and SEVERE hypoxic-acidemic insult. Acid-base balance was sampled and fetal ECGs continuously recorded. AC/DC were calculated: (1) for a spectrum of T values (T = 1÷50 beats; the parameter limits the range of oscillations detected by PRSA); (2) on entire series of fetal RR intervals or on “stable” series that excluded FHR decelerations caused by UCOs. Results AC and DC progressively increased with UCOs phases (MILD vs. MODERATE and MODERATE vs. SEVERE, p<0.05 for DC  = 2–5, and AC  = 1–3). The time evolution of AC/DC correlated to acid-base balance (0.4<<0.9, p<0.05) with the highest for . PRSA was not independent from FHR decelerations caused by UCOs. Conclusions This is the first in-vivo evaluation of PRSA on FHR analysis. In the presence of acute hypoxic-acidemia we found increasing values of AC/DC suggesting an activation of ANS. This correlation was strongest on time scale dominated by parasympathetic modulations. We identified the best performing parameters (), and found that AC/DC computation is not independent from FHR decelerations. These findings establish the basis for future clinical studies.

Monitoring fetal electrocortical activity during labour for predicting worsening acidemia: a prospective study in the ovine fetus near term.

Background Severe fetal acidemia during labour with arterial pH below 7.00 is associated with increased risk of hypoxic-ischemic brain injury. Electronic fetal heart rate (FHR) monitoring, the mainstay of intrapartum surveillance, has poor specificity for detecting fetal acidemia. We studied brain electrical activity measured with electrocorticogram (ECOG) in the near term ovine fetus subjected to repetitive umbilical cord occlusions (UCO) inducing FHR decelerations, as might be seen in human labour, to delineate the time-course for ECOG changes with worsening acidemia and thereby assess the potential clinical utility of fetal ECOG. Methodology/Principal Findings Ten chronically catheterized fetal sheep were studied through a series of mild, moderate and severe UCO until the arterial pH was below 7.00. At a pH of 7.24±0.04, 52±13 min prior to the pH dropping <7.00, spectral edge frequency (SEF) increased to 23±2 Hz from 3±1 Hz during each FHR deceleration (p<0.001) and was correlated to decreases in FHR and in fetal arterial blood pressure during each FHR deceleration (p<0.001). Conclusions/Significance The UCO-related changes in ECOG occurred in advance of the pH decreasing below 7.00. These ECOG changes may be a protective mechanism suppressing non-essential energy needs when oxygen supply to the fetal brain is decreased acutely. By detecting such “adaptive brain shutdown,” the need for delivery in high risk pregnant patients may be more accurately predicted than with FHR monitoring alone. Therefore, monitoring fetal electroencephalogram (EEG, the human equivalent of ECOG) during human labour may be a useful adjunct to FHR monitoring.