Human plasma inhibits arachidonic acid metabolism and platelet aggregation. This helps human form a haemostatic control system that prevents the progress of certain aggregatory or inflammatory reactions. Whether this property of plasma is unique to human or extends to other species is not well known. It is speculated that this protective ability of plasma remains evolutionarily conserved in different mammals. In order to confirm this, the effect of plasma from 12 different mammalian species was investigated for its inhibitory potential against arachidonic acid metabolism and platelet aggregation. Metabolism of arachidonic acid by cyclooxygenase and lipoxygenase pathways was studies using radio-immuno assay and thin layer chromatography while platelet aggregation in the plasma of various mammals was monitored following turbedmetric method in a dual channel aggregometer. Results indicate that inhibition of AA metabolism and platelet aggregation is a common feature of plasma obtained from different mammalian species, although there exists large interspecies variation. This shows that besides human, other mammals also possess general protective mechanisms against various aggregatory and inflammatory conditions and this anti-inflammatory property of the plasma is evolutionarily conserved in mammalian species. The most likely candidates responsible for these properties of plasma include haptoglobin, albumin and lipoproteins. Aspirin- a prototype of non-steroidal anti-inflammatory drugs exert its anti-inflammatory and antiplatelet effects by irreversibly inhibiting COX. Therefore, inhibition of COX and platelet aggregation is an important therapeutic target for treating a number of inflammatory and cardiovascular diseases  in the presence and absence of test plasma. The reaction was allowed to proceed with gentle shaking at 37 °C for 15 min. The reaction was stopped by adding ethyl acetate (7 mL) and citric acid (0.4 mL of 0.4 M). Again centrifugation was done at 4 °C to separate the organic layer. This organic layer was evaporated to dryness using liquid nitrogen. The residual material was applied (20 µL) to thin layer chromatography plates containing silica gel. The standards of all the metabolites such as 12-hydroxy-eicosatetraenoic acid (HETE)-a stable metabolite of 12-HpETE, lipoxygenase product 1 (LP1), TXB2 (a more stable metabolite of TXA2), and AA were separately plotted. The plates were developed in two different solvent systems. Various lipoxygenasse products such as 12-HETE were separated using acetic acid/ether/petroleum ether  solvent system while ethyl acetate/iso-octane/water/acetic acid  solvent system was used to separate cyclooxygenase products. Finally radioactive zones were located and quantified.AA metabolism was studied using the methods as described previously ,34. In bp < 0.05 was considered statistically significant.Data are represented as the mean ± SEM unless stated otherwise. Statistical significance was assessed by student t test. In conclusion, the conservation of antiplatelet and dual LOX and COX inhibitory activities of plasma across various mammalian species indicates that this phenomenon probably forms a part of a haemostatic control system that could help to curtail the progress of certain aggregatory or inflammatory reactions. The fact that these anti-inflammatory activities of plasma are conserved across various species also suggests that there might be some common plasma components responsible for these observed effects. The most important candidates for these unique properties of plasma are albumin, haptoglobin and plasma lipoproteins.


The ultrasound-guided proximal intercostal block (PICB) is performed at the proximal intercostal space (ICS) between the internal intercostal membrane (IIM) and the endothoracic fascia/parietal pleura (EFPP) complex. Injectate spread may follow several routes and allow for multilevel trunk analgesia. The goal of this study was to examine the anatomical spread of large-volume PICB injections and its relevance to breast surgery analgesia.Fifteen two-level PICBs were performed in ten soft-embalmed cadavers. Radiographic contrast mixed with methylene blue was injected at the 2nd(15 ml) and 4th(25 ml) ICS, respectively. Fluoroscopy and dissection were performed to examine the injectate spread. Additionally, the medical records of 12 patients who had PICB for breast surgery were reviewed for documented dermatomal levels of clinical hypoesthesia. The records of twelve matched patients who had the same operations without PICB were reviewed to compare analgesia and opioid consumption.Median contrast/dye spread was 4 (2–8) and 3 (2–5) vertebral segments by fluoroscopy and dissection respectively. Dissection revealed injectate spread to the adjacent paravertebral space, T3 (60%) and T5 (27%), and cranio-caudal spread along the endothoracic fascia (80%). Clinically, the median documented area of hypoesthesia was 5 (4–7) dermatomes with 100 and 92% of the injections covering adjacent T3 and T5 dermatomes, respectively. The patients with PICB had significantly lower perioperative opioid consumption and trend towards lower pain scores.In this anatomical study, PICB at the 2nd and 4th ICS produced lateral spread along the corresponding intercostal space, medial spread to the adjacent paravertebral/epidural space and cranio-caudal spread along the endothoracic fascial plane. Clinically, combined PICBs at the same levels resulted in consistent segmental chest wall analgesia and reduction in perioperative opioid consumption after breast surgery. The incomplete overlap between paravertebral spread in the anatomical study and area of hypoesthesia in our clinical findings, suggests that additional non-paravertebral routes of injectate distribution, such as the endothoracic fascial plane, may play important clinical role in the multi-level coverage provided by this block technique. Regional anesthesia has been consistently associated with superior pain control, lower opioid use and related side effects, when compared to conventional opioid-based analgesia –3. AppliThe intercostal space (ICS) communicates proximally  with the paravertebral space - as little as 1 ml dye injected into the ICS can spread to the paravertebral space . A largeThe goals of this study were to examine the anatomical spread of PICB injectate and explore its translation into clinical analgesia after breast surgery. The anatomy part of the study assessed the spread of methylene blue and radiographic contrast injection into the IIM-EFPPC plane of cadavers with both fluoroscopy and anatomical dissection. The clinical part consisted of a retrospective medical records review of patients who had undergone breast surgery under general anesthesia (GA) with and without PICB, examining the dermatomal analgesia/hypoesthesia distribution and the analgesic effect of the PICB.After IRB review and exemption, ten cadavers were prepared for the study by soft embalming technique . The cadThe injectate was prepared by mixing a radiographic contrast agent  30 ml with methylene blue 2 ml and diluted with water to 80 ml. After the needle was in satisfactory position by ultrasound imaging, 15 ml of injectate was injected at the 2nd proximal ICS and 25 ml at the 4th proximal ICS over 1–2 min. Real-time fluoroscopy was performed and recorded immediately after each injection to evaluate the spread of contrast  and PICB retrospectively, in order to compare the documented dermatomal levels of analgesia and hypoesthesia with the block to the results of the anatomical study. As the PICB technique had been introduced to our institution shortly before our study, the effects of the blocks, including dermatomal spread, were being assessed and documented in great detail for quality assurance. In order to compare the analgesic effect to the GA group, we performed sample size calculations, aiming to detect a 50% decrease of pain scores in the PICB group. Kim et al.  showed aThe PICBs were performed using a SonoSite X-Porte US machine with a linear 38 mm 10–12 MHz ultrasound probe  and the 21G 80 mm Sonoplex needle . Blocks were performed with standard ASA monitoring. The scanning and needling techniques were identical as in the anatomical study Fig. . Once thThe research team matched other 12 patients who had had the same operation with the same surgeon under general anesthesia without blocks to compare pain scores and opioids consumption. The statistical analysis included T test for normal distribution and Mann-Whitney U test for non-normal distribution, utilizing PASW statistics software (SPSS) 18.0 .All patients received general anesthesia . The medication choices were at the discretion of the anesthesiologist including administration of perioperative muscle relaxant, sedative and analgesics. Recorded perioperative opioid administration included all opioids given in the pre-, intra-, and post- operative periods up until discharge from the recovery room, converted to mg morphine equivalent (MME) IV units.PICB injections were performed in 10 cadavers. Two level injections at 2nd and 4th ICS were performed in 15 chest walls.  or TPVBs were excluded). Demographic data and injectate spread interpretation are shown in Table T2 and T4 levels were covered 100% by intercostal spread, by both fluoroscopy and dissection. However, adjacent T3 paravertebral/epidural spread was 53% (fluoroscopy) and 60% (dissection), whereas adjacent T5 level coverage was 67% (fluoroscopy) and 27% (dissection)  of the dissections showed methylene blue staining of the endothoracic fascia at least from 2nd to 5th ICS, without any staining of the visceral pleura Fig. . Three sThe average distances from midline (spinous processes) to needle entry points were 4.35+/− 1.06 cm at the 2ndICS and 3.8+/− 1.13 cm at the 4thICS. The average depth  was 2.01+/− 0.56 cm at the 2ndICS and 1.72+/− 0.40 cm at the 4thICS. The average needle visualization, by needle visualization score was fair. .The demographic data and the dermatomal hyposesthesia/analgesia distribution in the 12 patients who underwent breast surgeries with PICB are presented in Table The documented median hypoesthesia area was 5 dermatomes (range 4–7 dermatomes) and the distribution is shown in Fig. P values = 0.63, 0.11, 0.57 and 0.14 respectively).Table The comparison of pain scores and opioid consumption between 12 patients receiving PICB and general anesthesia (GA) and 12 matched patients receiving GA alone (same operation performed by the same surgeon), is presented in Table Truncal regional anesthesia techniques such as TPVB and the classic intercostal blocks have been utilized for anesthesia and/or analgesia for patients undergoing breast surgery , 4, 5. RThe proximal portion of the ICS  contains the intercostal nerves and communicates with the paravertebral space medially. Paraskeuopoulos et al. have demonstrated that as little as 1 ml methylene blue injected into the ICS 5 cm lateral to the spinous processes can spread to the paravertebral space . TherefoAs the breast is mainly innervated by T2-T5 spinal nerves  and the th intercostal space) but demonstrated great variability in paravertebral spread (0–7 segments), similar to the variability of paravertebral spread in TPVB described in previous studies [The PICBs produced consistent distribution within the injected intercostal space  through the potential space between the pleura and the internal intercostal muscle, which supports this hypothesis. The three dissections which revealed no endothoracic or adjacent ICS spread, but extensive paraspinal muscle staining might be explained with inadvertently shallow needle placement causing injectate spread into muscle instead of endothoracic fascia plane. Predictable 2nd and 4th intercostal distribution combined with paravertebral and endothoracic fascia plane spread may present a plausible complex model for reliable dermatomal coverage of PICB in the clinical finding. The multiple anatomical routes of injectate distribution with PICB, influenced particularly by the block needle tip position relative to the internal intercostal membrane, may provide possible explanations to the inter-individual variability in segmental spread and ultimately, in clinical coverage.Our dissections revealed methylene blue spread inside the respective intercostal spaces and along the investing tissues around the injection sites in 80% of the specimens. The endothoracic fascia is interposed between the parietal pleura and the superior costotransverse ligament and extends laterally as an intervening fascia between pleura and internal intercostal membrane. The absence of dye on the visceral pleura and the underlying lung surface Fig.  suggestsPotential advantages of the PICB over TPVB , include superior US-visualization of pleura and block needle due to shorter skin-to-target distance and more perpendicular US beam-to-pleura/needle orientation (unpublished data). Additionally, the longer distance of block needle from spinal canal may hypothetically convey improved safety, especially in patients who are at increased risk of bleeding complications.Our clinical findings suggest that high-volume two-level PICBs consistently produce sensory block in dermatomes relevant to adequate analgesia after breast surgery, and could logically decrease pain and opioid consumption after mastectomy and lumpectomy. The surprisingly low median pain scores on arrival to recovery room in both groups are likely due to a combination of residual general anesthetic effect, the effect of other analgesics administered in the operating room and even individual pain thresholds. Our study was not designed and powered to examine differences in pain scores and only demonstrated a trend towards lower pain scores in the PICB group. As the shortcomings of our clinical study stem from its retrospective design with no anesthetic/analgesic standardization, well-controlled prospective trials are needed to further evaluate the analgesic, anesthetic and recovery profiles of PICB.The discrepancy between the observed segmental spread by fluoroscopy  and dissection  may also seem surprising. Among the logical explanations, two appear most plausible:  while thLarge-volume ultrasound-guided proximal intercostal blocks, performed at the 2nd and 4th intercostal spaces, produced a predictable lateral injectate spread along the corresponding intercostal neurovascular bundle, a less consistent medial spread to the adjacent paravertebral/epidural spaces and a contiguous endothoracic fascia plane distribution in the anatomy study. The incomplete overlap of anatomical paravertebral spread and dermatomal distribution of clinical hypoesthesia suggests additional non-paravertebral route of injectate spread, including the endothoracic fascia plane, confirmed by the staining patterns in the anatomy specimens.


Acute hepatitis E virus infections occurred during the third trimester in 2 pregnant women in France who sought treatment with nonspecific symptoms or asymptomatic elevation of liver enzymes. Infection cleared quickly in both women. We detected no hepatitis E RNA in 1 newborn’s feces at 3 weeks of age. In low-income countries, estimates of 20 million hepatitis E virus (HEV) infections and 70,000 deaths have been reported for 2005. Increased mortality rate in pregnant women is well described but unexplained; pregnant women with symptomatic HEV infection have a 10-fold higher probability of death, especially in countries of the Indian subcontinent and Africa in which genotypes 1 and 2 are prevalent  . HepatitThe second case-patient was a 38-year-old woman with intrauterine growth restriction found at 24 weeks’ gestation. Increased ALT at 30 weeks’ gestation led to HEV diagnosis with detectable HEV IgM and viremia (2.98 log IU/mL). In retrospective testing, we detected HEV RNA in blood (2.37 log IU/mL) but not in the amniotic fluid at the time of amniocentesis (27 weeks’ gestation). HEV genotyping was unsuccessful. The patient’s liver abnormalities resolved by 33 weeks’ gestation. We performed no fetal or neonatal monitoring. The woman delivered a low-weight  but otherwise healthy baby at 38 weeks’ gestation. The maternal physical examination remained normal in the weeks after delivery; we performed no further hepatitis follow-up. Both patients had nonspecific symptoms or asymptomatic ALT elevation despite acute HEV infections in the third trimester of pregnancy. The rapid decline in ALT suggested a rapid clearance of infection in both. The lack of HEV RNA in the feces of 1 newborn indicated that infection was not transmitted; the lack of detectable RNA in the amniotic fluid suggested the same in the second case. We cannot rule out fetal HEV infection in patient 2 after 27 weeks’ gestation, even though such an infection is unlikely because both children were born healthy. These mild courses of illness are similar to previous reported outcomes involving HEV genotype 3 (HEV infection among pregnant women in industrialized countries is rarely reported, perhaps because it is rare in this setting, or infection may be underdiagnosed because symptoms are mild or nonspecific. Indeed, no acute infection or seroconversions were observed among pregnant women in a prospective study in France (


Cytochrome-c-oxidase (CCO) plays a central role in mitochondrial oxidative metabolism and ATP synthesis. Using a novel broadband NIRS system, we investigated the impact of pressure passivity of cerebral metabolism (CCO), oxygenation (haemoglobin difference (HbD)) and cerebral blood volume ) in 23 term infants following HIE during therapeutic hypothermia (HT). Sixty-minute epochs of data from each infant were studied using wavelet analysis at a mean age of 48 h. Wavelet semblance (a measure of phase difference) was calculated to compare reactivity between mean arterial blood pressure (MABP) with oxCCO, HbD and HbT. OxCCO-MABP semblance correlated with thalamic Lac/NAA . OxCCO-MABP semblance also differed between groups of infants with mild to moderate and severe injury measured using brain MRI score (p = 0.04), thalamic Lac/NAA (p = 0.04) and neurodevelopmental outcome at one year (p = 0.04). Pressure passive changes in cerebral metabolism were associated with injury severity indicated by thalamic Lac/NAA, MRI scores and neurodevelopmental assessment at one year of age.Hypoxic ischemic encephalopathy (HIE) leads to significant morbidity and mortality. Impaired autoregulation after hypoxia-ischaemia has been suggested to contribute further to injury. Thalamic lactate/N-Acetylasperate (Lac/NAA) peak area ratio of > 0.3 on proton ( These metabolic changes have been termed secondary energy failure. These MRS studies were important for the concept that interventions such as cooling ameliorate the subsequent secondary energy failure.69 Currently, thalamic lactate/NAA peak area ratio acquired between day 5 and 14 predicts outcome in HIE using a cut off threshold of 0.3;10 this ratio is used with conventional MRI for counselling and prognosis.11Intrapartum hypoxic-ischemic injury leading to hypoxic ischaemic encephalopathy (HIE) is a significant cause of neonatal morbidity and mortality. Each year across the world, approximately 1 million babies die following intrapartum complications.12 and monitoring cerebral autoregulation following HIE,1317 but direct assessment of cerebral mitochondrial function in relation to cerebrovascular reactivity has not been investigated so far.There has been recent interest in the use of blood biomarkers18 Using a broadband NIRS system, concentration changes in the oxidation state of CCO (oxCCO) can be measured along with changes in oxy- and deoxy-haemoglobin (HbO2 and Hb), with derived changes in haemoglobin difference (HbD=HbO2−Hb) and total haemoglobin ((HbT=HbO2+Hb).19 Changes in [oxCCO] indicate the status of the mitochondrial function and has been used to monitor the cerebral energy state following HIE in preclinical21 and clinical studies.2224Cytochrome-c-oxidase (CCO) is the terminal electron acceptor inside the mitochondrial electron transport chain (ETC). It plays a crucial role in mitochondrial oxidative metabolism and is responsible for more than 95% of ATP synthesis.2527 In recent years, further attempts have been made to examine the cerebrovascular reactivity status of the brain and its relationship with outcome in neonates with HIE who underwent therapeutic hypothermia, using NIRS-based haemodynamic indices.1316 A close relationship between dis-autoregulation and abnormal cerebral metabolism has been described in adults after traumatic brain injury but pressure passivity was not related to CBF.28Cerebral autoregulation (CA) maintains a constant cerebral blood flow (CBF) over a range of cerebral perfusion pressure and protects the brain from hypo- and hyperperfusion. Cerebrovascular circulatory function is controlled through neural, myogenic and metabolic mechanisms. Cerebral vasoparalysis leading to abnormal cerebral haemodynamics and impaired CA following HIE was associated with poor outcome in pre-hypothermic era.8 Both preclinical and clinical studies using phosphorus magnetic resonance spectroscopy (31P MRS) have demonstrated the depletion in cerebral energy state immediately after the HI insult (primary energy failure) followed by a further phase of deterioration 6–24 h after HI (secondary energy failure). During this secondary phase, phosphocreatinine (PCr) and neucleotide triphosphate (NTP) fell and inorganic phosphate increased (Pi) despite maintenance of adequate oxygenation and circulation. The secondary phase marked by the onset of seizures, cytotoxic oedema, accumulation of cytokines and mitochondrial failure that leads to further call death.29 The degree of energy failure influences the type of cell death.31 These findings further raise the importance of reviewing the relationship of a metabolic reactivity index with outcome following HIE. A cot side metabolic reactivity index using CCO has never been examined before. This is particularly intriguing in the current era, in view of the influence of HT on other early prognostic biomarkers. The predictive ability of amplitude integrated EEG32 and the neurological examination at 72 h are influenced by hypothermia.33 Thoresen and coworkers35 also demonstrated that the cerebral resistance index (RI) on Doppler ultrasound has lost the predictive value during HT. In the pre-HT era, resistance index (RI) < 0.55 was found to be a predictor of adverse outcome at 18 months following HIE in 84% of normothermic infants while it predicted outcome in only 60% on infants on day 2 during HT.35Disturbances in cerebral oxidative metabolism following HIE are well documented.37 Both transcranial Doppler and NIRS-derived reactivity indices examined the relationship between spontaneous slow wave oscillations (0.003–0.05 Hz) in mean arterial blood pressure (MABP), Doppler flow velocity,37 cerebral blood volume (CBV)16 and cerebral oxygenation3842 in the time domain. Similarly, coherence and gain have been used in frequency domain analysis.43 One of the major limitations for these techniques is the assumption of stationary relationship between the variables. Cerebrovascular autoregulation is dynamic, nonstationary and the signals vary both in time and frequency, more under pathological conditions.44 Wavelet-based analysis can overcome this issue and characterise autoregulation with improved time–frequency resolution following brain injury both in adults46 and the newborn.17Several methodologies have been used to assess cerebrovascular reactivity in both term and preterm infants.1H MRS, (b) will be able to identify infants with severe HIE, and (c) will be able to differentiate the infants with severe HIE from the group with mild to moderate HIE based on both early biomarker and neurodevelopmental assessment at one year of age.We hypothesised that a metabolic reactivity index based on wavelet analysis of slow wave (SW) oscillations of oxCCO and MABP measured at 48 h after birth – (a) will correlate with thalamic lactate/NAA peak area ratio on We aimed to investigate the effects of disturbances in brain metabolism following HIE on outcome, using a metabolic reactivity index derived from wavelet analysis between oxCCO and MABP in a cohort of infants undergoing hypothermia (HT) following HIE.This prospective observational study (Baby Brain Study) was approved by the Research Ethics Committee (REC) of University College London Hospital and London Bloomsbury REC (reference: 13/LO/0106) in accordance with the declaration of Helsinki. Written informed consent was obtained from parents before each study.47 This diagnosis was confirmed by clinical examination, umbilical cord gas acidosis and abnormal electrical activity on electroencephalogram (EEG) or amplitude-integrated electroencephalogram (aEEG). A servo-controlled cooling machine  was used to maintain the temperature at 33.5℃ for 72 h before gradually increasing the temperature to 37℃ over 14 h.Stable term infants admitted to the neonatal unit in University College London Hospital for HT following HIE were eligible for the study. Unstable and sick infants or infants with congenital abnormalities were excluded from the study. Clinical decisions regarding the care of the infant were taken by the intensive care team in line with the local and national guidelines. Whole body HT with intracorporeal temperature monitoring was instituted in infants with evidence of moderate to severe HIE as early as possible after birth as per the National Institute for Health and Clinical Excellence (NICE) guidance.Physiological and broadband NIRS data were collected over 2–4 h, from which a 60-min period from each infant during periods of clinical stability was selected for this analysis. All infants were sedated (continuous intravenous infusion of morphine sulphate), muscle-relaxed (atracurium intravenous infusion) and ventilated during HT. Invasive blood pressure recording was collected continuously from indwelling umbilical arterial catheter. Physiological data from individual patient monitors  were captured using ixTrend software , down-sampled and synchronised with broadband NIRS timeframe using a MATLAB -based software using spline interpolation. NIRS data were collected using a customised software developed in LabView . 2224 which is capable of monitoring Δ[oxCCO] as well as Δ[HbO2] and Δ[HHb] in the neonatal brain with improved signal quality measured over 136 wavelengths. The system consists of an optical fibre illuminator  with a stable white light source. The source is coupled to two optical fibre bundles which illuminate the tissue. Four detector bundles collect the attenuated light emerging from the tissue at increasing distances from each source . The optical fibres are held to the tissue in a custom 3D printed holder. At the detection end, a lens-based spectrometer  and a front-illuminated CCD camera  resolve the intensity spectrum across 770–906 nm for the eight detectors simultaneously. The longest source-detector distance of 3 cm was chosen to ensure an optimal depth penetration and the differential path length (DPF) of 4.99 was used48 to calculate the concentration changes of different chromophores using UCLn algorithm.19CCO contains four active metal redox centres; one of them, the CuA is a dominant near-infrared (NIR) chromophore and the primary contributor for the NIR spectral signature. Detection of CCO using NIRS is more difficult than other chromophores as its in-vivo concentration is less than 10% of that of haemoglobin and has a broad spectral signature. Broadband NIRS can accurately resolve the spectral changes due to oxCCO without crosstalk from the haemoglobin chromophores. We have recently developed a new broadband NIRS system,49 After artefact removal, NIRS data were processed with an automatic wavelet de-noising function in MATLAB to reduce the high frequency noise but maintain the trend information.MABP and NIRS data were visually inspected for any artefacts. Sudden changes in NIRS variables greater than 15% from baseline and not consistent over all the signals were identified as artefacts. Brief transient artefacts in MABP were removed by simple interpolation. Artefacts in NIRS data were removed by using moving standard deviation and spline interpolation in MATLAB.5054 to be a powerful mathematical tool for time-frequency analysis for both stationary and non-stationary time series. Our group have used this technique in multiple studies55 including a recent study to illustrate and characterise changes in cerebrovascular reactivity following adult brain injury.46 Tian et al.17 has also suggested a potential clinical use of this technique to assess the dynamic CA following HIE.Keeping in mind the non-stationary aspect of CA, SWs in MABP and NIRS signals were analysed using wavelet-based techniques to achieve high time-frequency resolution. The continuous wavelet transform (CWT) with the complex Morlet wavelet has been shown46 to determine wavelet coherence and semblance. Wavelet coherence based on CWT was calculated as a measure of similarity in spectral power and dynamic relationship between spontaneous oscillations in MABP and NIRS variables (oxCCO and HbD). Wavelet coherence varies from 0 to +1 depending on the strength of relationship between the variables. Wavelet semblance was calculated as a measure of instantaneous phase difference and creates an index from + 1  to −1 . Wavelet semblance bears a similarity to previously described time-domain indices 57 and gives us the opportunity to assess the cerebrovascular and cerebral metabolic reactivity in a similar fashion.We have used the same MATLAB-based tools described by Highton et al.p < 0.05.The haemodynamic (semblance of MABP and HbD or HbT) and metabolic (semblance of MABP with oxCCO) reactivity indices were calculated across a 60-min study period for each infant and the mean values were used for comparison. Indices and variables were documented using median, range or with mean ± standard deviation as appropriate. Datasets were checked for normality using D’Agostino-Pearson omnibus normality test before further statistical analysis in Graphpad Prism 6 . Welch’s correction was performed while comparing between groups when standard deviation was different. Statistical significance was considered as 58 Thalamic Lac/NAA peak area ratio obtained from 1H MRS is a robust quantitative measurement within the neonatal period for prediction of neurodevelopmental outcome following HIE.10 Lac/NAA < 0.3 indicated good motor outcome following HIE in this systematic review and meta-analysis. We have used the NICHD neonatal MRI brain injury scoring system described by Shankaran et al.11 for prediction of neurodevelopmental outcome at six to seven years of age following HIE. Specific patterns of MRI brain injury 2B , anterior or posterior limb of internal capsule (ALIC or PLIC), or watershed (WS) infarction and cerebral lesions) and three  in this study were highly predictive of death or IQ < 70 at six to seven years of age.MRI of brain is the imaging modality of choice following HIE and together with MRS, clinically used to assess the injury severity and for prognostication.1H MRS were performed between day 5 and 7 using a 3 T Philips MRI scanner . T1-weighted imaging was acquired using an inversion-recovery prepared spoiled gradient echo . T2-weighted imaging was acquired using 2D fast spin echo , Diffusion tensor imaging was acquired with 32 directions of diffusion weighting with b-values of 0 and 750. Apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps were reconstructed inline on the scanner. For MRS, a single PRESS voxel of 1.5 × 1.5 × 1.5 cm was positioned to encompass as much of left thalamus as possible while avoiding overlap with CSF . A dynamic series of 16 subspectra were acquired, each with eight averages. These subspectra were subsequently and individually frequency and phase corrected before summation to yield the final full spectrum.59 This methodology allows for any subspectra corrupted by patient motion to be removed from the final summation. NAA, choline, creatine and lactate peaks were identified at 2.02, 3.02, 3.24 and 1.33 ppm, respectively. Spectra were fitted using AMARES60 as implemented in the jMRUI magnetic resonance software package.61MRI and A paediatric neuroradiologist (RG) scored all MRI images.All infants born in our hospital had regular neurodevelopmental follow-up and were assessed with Bayley Scales of Infant Development-III. Infants born in other hospitals and were transferred to us for management of HIE were also offered the first assessment in our hospital and had the opportunity to choose further assessments in either our hospital or in their local units. A score of <85 was considered adverse outcome . In this study, infants with adverse neurodevelopmental outcome and death were compared with the group who survived with a good outcome at one year of age.63 Power spectral density (PSD) analysis was performed using Welch’s method to determine the power in the SW range and a power index calculated based on SW power/Total power using MATLAB.MABP variability in the SW spectrum (0.003–0.05 Hz) and the relationship with outcome biomarkers were reviewed in the study population. It was important to identify whether MABP variability was directly related to outcome in this cohort and might have influenced the relationship of metabolic reactivity index from wavelet analysis with outcome. This was performed in the frequency domain using established spectral analysis techniques.2 (tcCO2) remained stable. All infants had MRI and MRS of brain between day 5 and 7 of life. Four infants died either in the neonatal period or within the first year of life. Patient characteristics are presented in 2) and MABP) were noted during the study periods.Twenty-three term newborn infants with moderate to severe HIE participated in the study while undergoing HT. Sixty-minute datasets collected during stable periods at a mean age of 48 h were analysed during which transcutaneous COWavelet coherence and semblance between oxCCO and MABP were calculated as 0.37 ± 0.08 and 0.06 ± 0.13, respectively. HbD-MABP wavelet coherence and semblance were 0.42 ± 0.09 and 0.12 ± 0.17, while HbT-MABP coherence and semblance were 0.38 ± 0.06 and 0.07 ± 0.14. Thalamic Lac/NAA on MRS varied between 0.11 and 2.64.r = 0.48, r2 0.23, p = 0.02)     . oxCCO-M = 0.62) . The relp 0.04     0.94) between the groups (OxCCO-MABP semblance compared between two groups based on MRI score (MRI score < 2B and ≥ 2B) demonstrated a significant difference  1.52)  (two tailed d) 1.52) .A strength of wavelet analysis is the possibility to investigate the relationships between MABP and NIRS indices in detail for individual studies. p value 0.26 and 0.93 respectively, Mann–Whitney test) between groups of infants with normal or poor outcome .The power spectral density analysis of MABP waveform was performed in all infants. Average power in the SW frequency range (0.003–0.5 Hz) with SW/total power index was calculated. No significant differences were noted  had better outcome compared to infants with disturbed metabolic reactivity (high wavelet semblance 0 to +1). Variability of MABP itself did not influence the relationship of oxCCO-MABP semblance with outcome. OxCCO-MABP wavelet coherence as well as other wavelet indices between MABP and haemoglobin-based indices (HbD and HbT) did not correlate with outcome biomarkers and did not differ among groups of newborn infants with death or poor outcome and normal outcome. Metabolic reactivity index (oxCCO-MABP semblance) appears to be a promising cot side indicator of outcome following HIE.53 This corroborates with our findings of oxCCO-MABP wavelet semblance being a superior index of reactivity compared to wavelet coherence.OxCCO-MABP semblance is likely to reflect the metabolic response to blood flow changes and substrate delivery to mitochondria and as a result becomes a marker of metabolic reactivity. Our findings suggest that oxCCO-MABP semblance can identify those neonates where impaired CA is compromising flow-metabolism coupling. Therefore, it is related to outcome more closely than HbD and HbT indices from wavelet analysis. Wavelet coherence indicates where signal powers vary together between the variables and reflects change in power and synchronisation of phase. In contrast, wavelet semblance gives a measure of phase difference, making it a more effective index for assessment of cerebral reactivity and autoregulation. The phase dynamics derived from wavelet analysis between MABP and cerebral blood flow velocity (CBFV) reflected most of the linear and non-stationary characteristics of CA in a study by Latka et al.64 Pressure passivity of the cerebral circulation has been documented in sick term16 and preterm29 infants and has been related to outcome in both newborns14 and adults.65 Recent studies in newborn term infants following perinatal brain injury have suggested the importance of early identification of loss of autoregulation indicating the need for continuous monitoring with appropriate indices of cerebrovascular reactivity.1317CA in the newborn brain attempts to maintain a constant CBF over a range of perfusion pressure and a key protective mechanism.66 but this relationship is likely to be disturbed following HIE. Cerebral nitric oxide (NO) maintains cerebrovascular tone by modulating CBF.67 Under normal conditions, intra-mitochondrial Ca++ activates mitochondrial phosphates, which in turn activates cytochrome c and CCO. Following HIE, increased Ca++ influx inside cell activates neuronal nitric oxide synthase (nNOS) stimulating the production of NO from L-arginine and oxygen.68 NO disrupts the mitochondrial respiratory chain by impairing the function of CCO (complex 4) and complex 1 and induce apoptosis.6973Metabolic regulation of blood flow is well established. CBF and cerebral metabolism are tightly controlled in the healthy brain,NO-mediated injury pathways may explain our findings of passive (zero to +1) cerebral metabolic reactivity (oxCCO-MABP semblance) in infants with severe perinatal brain injury and poor outcomes. It is likely that increase in cerebral NO production following hypoxic ischaemic injury is responsible for the haemodynamic changes as well as the secondary energy failure by inhibition of mitochondrial respiration at the level of CCO.75 This second phase of increased cerebral perfusion is related to the secondary energy failure,76 during which an increase in extracellular concentration of citrulline77 has been documented in late gestation fetal sheep. Citrulline is produced during the production of NO from L-arginine by NOS, suggesting an increased production of NO during this period. NO induces cerebral vasodilation as well as neuronal death through free radical injury. Relationship of oxCCO-MABP semblance at 48 h with outcome most likely reflects the effect of established mitochondrial injury and vasoparasis at this point. It is interesting to note that inhibiting NOS following cerebral ischaemia in fetal sheep increased cerebral injury,78 most likely by limiting the substrate delivery to ETC within the already compromised mitochondria.80Two periods of vasoparasis have been identified following HIE. The initial phase happens soon after the hypoxic ischaemic insult and the second phase 12–24 h later, which continues for hours or days depending on the injury severity.81 Xenon-133 clearance,82 (positron emission tomography (PET),83 single photon emission computed topography (SPECT)84 and perfusion-weighted MRI.85 But these techniques are not suitable for continuous monitoring at cot side in the neonatal intensive care. NIRS has emerged as an alternative tool for monitoring cerebrovascular reactivity at the cot side.1317 Identification of an optimal blood pressure was examined in a group of infants with HIE to find the optimal vasoreactivity.16 Infants with significant injury following HIE spent a greater proportion of time below optimal mean arterial blood pressure (MAPOPT) and had a greater deviation from MAPOPT compared to infants with normal outcome. Time spent below MAPOPT was also associated with neurodevelopmental impairment at two years. Massaro et al.15 demonstrated a similar relationship using pressure passivity index (PPI), an indicator of duration of pressure passive circulation.15 These methodologies were based either in time or frequency domain. Several studies have looked at the cerebrovascular reactivity but the metabolic regulation or metabolic reactivity indices has long been overlooked.Monitoring cerebrovascular reactivity and assessing its relation to outcome has been an important focus following newborn brain injury. A series of different methodologies have been used to directly measure CBF – Doppler ultrasound,17 have demonstrated that using wavelet-based tools, it is possible to characterise CA better. Tian et al. described the time-scale-dependant nature of CA with demonstration of in-phase and antiphase coherence and their relationship to outcome following HIE. Wavelet semblance in this study examined the phase differences between MABP and NIRS indices. It is the cosine of the instantaneous phase difference and varies toward +1 when signals are closely aligned in phase and toward −1 when they are in antiphase. Wavelet semblance46 being in a scale of −1 to +1 can be used more intuitively, like time domain-based reactivity indices.86 The individual colour maps for wavelet coherence and semblance also help to understand the cerebral physiological changes over time in individual cases  and EEG are two different physiological measurements and a direct comparison between them is not possible based on our study, it is interesting to note that both wavelet approaches were able to differentiate between the poor and normal outcome groups after HIE.One of the major challenges for signal-processing for analysis of CA is the dynamic and non-stationary nature of cerebral slow-waves. Our group and recently Tian et al.al cases . Recentl2, arterial oxygen saturation, CBF, CBV and cerebral oxygen consumption. We have tried to keep these variables constant by choosing periods of clinical stability. We have measured transcutaneous CO2, SpO2 and other systemic variables continuously during the studies and selected 60-min epochs when these variables remained mostly stable with minimal changes. We also did not have the neurodevelopmental outcome for all infants and follow up at two years would be optimal, but it was encouraging to note the clear relationship between the metabolic reactivity index at 48 h and neurodevelopmental outcome at one year of age in our cohort.A significant proportion of infants admitted to our unit for management for HIE were transferred ex-utero from other local units. We could not obtain consent for study on day 1 for these infants as parents were not available. Findings of this study represent the effect of hypoxic ischaemic injury on cerebral metabolism at 48 h of life during HT. To interrogate the role of this metabolic reactivity index for understanding of the pathophysiological changes following HIE, measurements need to be taken early after hypoxia ischaemia and at regular intervals during HT and rewarming. Cerebrovascular reactivity can be affected by changes in pCOCerebral metabolic reactivity following HIE, as quantified by oxCCO-MABP semblance using wavelet analysis characterised and quantified cerebral metabolic changes in babies with HIE. This reactivity index, oxCCO-MABP semblance was associated with outcome biomarkers used for early prognostication of outcome after HIE as well as the neurodevelopmental outcome measured at one year of age. These findings support the feasibility of wavelet-based assessment of dynamic changes in cerebral metabolism and haemodynamics in newborn infants and the role of oxCCO-MABP semblance as a useful cot side biomarker to differentiate between the infants with good and poor outcome following HIE.


We report the full polyprotein genomic sequence of a West Nile virus strain isolated from Eurasian magpies dying with neurologic signs in Greece. Our findings demonstrate the local genetic evolution of the West Nile virus strain responsible for a human disease outbreak in the country that began in 2010. West Nile virus (WNV) is the etiologic agent of an ongoing human disease outbreak in Greece since 2010. Until 2014, successive yearly outbreaks occurred mainly in central and northeastern Greece (Pica pica), hooded crows (Corvus cornix), sparrows (Passer domesticus), and Eurasian collared doves (Streptopelia decaocto). Our team verified the presence of Eurasian magpies with neurologic signs in the area; affected birds were lethargic and unable to fly, stayed low to the ground, and had no reaction to external stimuli .In June 2017, one month before human cases occurred, dead wild birds were reported in the Argolida regional unit in the Peloponnese region of Greece. Through mid-July, local residents noticed a reduction of the native wild bird population, especially Eurasian magpies  ≈48 hours after infection, we transferred the flasks to −20°C for 4 hours. After thawing the supernatant and cells, we performed total RNA extraction using the PureLink RNA Mini Kit .We extracted brain tissue samples during necropsy for inoculation in Vero cell culture. We vortexed brain homogenates in phosphate-buffered saline and centrifuged them at 4,000 × https://www.thermofisher.com), followed by fragment separation with a 3730xl DNA Analyzer (Applied Biosystems). We verified all nucleotide changes from other WNV strains detected in the 8 positive WNV RNA culture extracts by PCR using the corresponding primers on the tissue extracts. We submitted the consensus sequence, obtained by alignment and assembling in MEGA version 7 software  showed that the Argolida-Greece-2017 strain had the highest sequence similarity (99.79%) to the Nea Santa-Greece-2010 strain  and 4 amino acid changes compared with the phylogenetically closer Nea Santa strain. Amino acid changes include the Ι159Μ in the envelope gene near the NYS glycosylation motif, the H22Y and A298V in the nonstructural (NS) 1 gene, and the K805R mutation in NS5 gene. We predicted that all amino acid changes in the polyprotein gene are tolerated in accordance with the Sorting Tolerant From Intolerant algorithm (In a recent study, Jiménez de Oya et al. performed experimental infection of Eurasian magpies with 2 WNV strains currently circulating in Europe; they found magpies to be highly susceptible to WNV infection, with low survival rates for both strains (Additional information about bird-virulent West Nile virus, Greece.


Overall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.The objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.For identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. Abstracts were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.A total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.Direct experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility. Perturbations in testicular development or function during fetal life may result in male reproductive disorders that present postnatally , which results in the development of testicular germ cell cancer (TGCC) in adulthood  in the human .Fetal Leydig cells are present in the interstitium of the testis from six GWs in humans and are responsible for the production of hormones involved in testicular descent. Insl3 is involved in the transabdominal phase of testicular descent, whilst testosterone is required to enable the testis to traverse the inguinal canal . Leydig in-vitro versus in-vivo), exposure regimen and drug metabolism, whilst also accounting for the impact of species differences for each of these parameters  to identify all experimental studies relating to testicular effects of fetal exposures to environmental, pharmaceutical and lifestyle factors, limited to studies utilizing human fetal tissues or cells. For identification of relevant epidemiological studies, we included the publications identified in a recent systematic review and meta-analysis of associations between prenatal exposures and male reproductive disorders , germ or somatic  cell development.We excluded studies according to the following criteria: exposure of tissues or cells representative of a period other than fetal life; exposure of non-testicular tissues or cells; outcomes other than those described above; and review articles.We searched the databases using a combination of medical subject headings and generic terms relating to effects of exposures on human fetal testis development . We idenin-vitro and ex-vivo (xenograft) approaches and results were assessed primarily for effects on testosterone secretion and on germ cell number (both expressed as % change compared to vehicle control). Effects on additional testicular hormones, AMH (produced by SC) and Insl3 (produced by LC) are also reported.Studies included The distribution of studies based on exposure type and year of publication is shown in Fig. n-butyl phthalate (DBP), which is metabolized to mono-n-butyl phthalate MBP. In 2015, DEHP was banned from general use under EU law under the REACH restriction  and has been replaced by di-isononyl phthalate (DiNP), and more recently di-isononyl cyclohexane-1,2-dicarboxylate (DINCH), both of which are deemed less biologically active  have used rodent models and have demonstrated a disruption of normal fetal testis development and subsequent development of male reproductive disorders, resulting from decreased fetal testosterone production in male offspring  and excreted . These sSeveral phthalate monoesters have been detected in maternal breast milk over a large concentration range (1.5–1 410 μg/L) and these have been correlated with infant hormone levels (n = 642), in comparison to non-exposed mothers with IBD (n = 1158)  and anti-epileptics  . This ca−8 M) and cord blood of newborns  has been associated with a significant reduction in AGD in male offspring (n = 366) (n = 168) and maternal DEHP metabolite, concentrations specifically during the first trimester, has also been described  at 3 months of age  testosterone production after 48 h   were dosed with 500 mg/kg MBP from ~7 to 15 weeks gestation and testicular and male reproductive effects were assessed in the neonate or adult. No effects on testicular morphology or germ cell number were identified at birth or adulthood following in-utero MBP exposure and testosterone levels at birth were unaffected. Importantly, there were no cases of hypospadias, cryptorchidism, impaired spermatogenesis or focal testicular dysgenesis in any of the exposed animals   using an organotypic culture system resulted in unchanged testosterone  production compared to control after 72 h of culture  to 10−5 M) in-vitro via an organotypic culture system resulted in an approximately 40-50% increase in apoptotic gonocytes compared to control testis tissue  for 72 h. Using a similar in-vitro system, MEHP-exposure resulted in a 40% reduction in germ cell number via increased apoptosis  at both 10−4 and 10−5 M MEHP under basal conditions, whilst 10−5 M MEHP resulted in a reduction in gonocyte number under basal and LH-stimulated conditions  with MEHP showed no effect on the mitotically quiescent germ cells  is also a component of epoxy-resins, used as the inner coating of metallic food and beverage cans. BPA has weak estrogenic properties, albeit several orders of magnitude less potent than endogenous 17β-oestradiol  resulted in a reduction of 20–50% , hLH- (30%) and hCG-stimulated (60%) conditions , whereas reduction in testosterone only occurred at the highest concentration (10−5 M) under hLH supplemented conditions  conditions  and lowest (10−8 M) concentrations of BPA tested and also only under basal conditions. However, levels of Insl3 were generally much lower in these basal conditions than in media supplemented with hLH or hCG. For gonadotrophin supplemented cultures, BPA did not affect Insl3 production at any of the concentrations tested, similar to the results already described for testosterone  in drinking water resulted in a modest (−19%) reduction in germ cells/mm2, indicating that a sustained increase in apoptosis of germ cells could reduce their number. Analysis of the individual germ cell populations demonstrated that there was a significant reduction in the proportion of germ cells expressing the gonocyte marker (AP2γ) with a reciprocal increase in pre-spermatogonial (MAGEA4) population. These findings are consistent with a reduction in gonocytes or alternatively an acceleration in the normal germ cell differentiation from gonocyte to pre-spermatogonium following BPA exposure  using the in-vitro experiments indicate the potential for BPA to reduce testosterone production from the fetal testis, this has been under basal conditions and xenograft studies have failed to demonstrate similar effects in either first or second trimester human fetal testis under gonadotrophin stimulation, as occurs in normal pregnancy. Furthermore, typical human exposure is likely to be well below the concentrations used for experimental studies involving animal(s) or human tissues based on meta-analysis and latest data on human BPA exposure using LC/MS/MS  are inconsistent. For experimental studies involving human testis tissue, whilst the results of Pesticides and fungicides represent a heterogeneous group of chemicals widely used in agriculture. Many of these agents  are known to have anti-androgenic properties . Whilst in-vivo studies demonstrating effects of in-utero exposure to the fungicides Procymidone  did not affect testosterone production under basal conditions; however, testosterone (−30%) and gene expression of the steroidogenic enzyme ‘steroidogenic acute regulatory protein’ (StAR) were significantly reduced under LH-induced conditions .Exposure to concentrations of Dieldrin relevant to environmental and maternal serum levels in pregnancy  the frequency of use during pregnancy may be high resulting in sustained exposures.Analgesics are the most commonly used medications worldwide. Several of these are available without prescription and it has been reported in many countries that 50–90% of women will use an analgesic at some stage during pregnancy (in-vivo and in-vitro approaches (in-vivo (20% reduction in AGD) (in-vitro (70% reduction in testosterone)   or in-vin in AGD , whilst The majority of studies that investigated associations between maternal analgesic exposure and cryptorchidism in male offspring have reported a significant positive association , which wSeveral recent studies have investigated the effect of analgesics on human fetal testis using experimental models Table .In-vitro studies using first trimester testis (8–12 GW) exposed to paracetamol (10−5 M) for 1–3 days did not alter testosterone production, compared with vehicle controls (−5 M)  or differences in the experimental system  into host castrate nude mice. Oral exposure of these mice to a human-relevant regimen of paracetamol  for 1 week significantly reduced (−45%) host serum testosterone and seminal vesicle (androgen dependent organ) weight (−18%), unlike a single daily exposure which had no effect on either parameter (The conflicting results between rodent and human −7 M to 10−4 M) was demonstrated  exposed to paracetamol (10−5 M) for 1–3 days found no alteration in germ cell number (in vitro study with a longer period (7 days) of exposure showed that similar paracetamol concentration (10−5 M) significantly reduced (−28%) gonocyte number  human fetal testis to aniline for 96 h had no effect on testosterone production across a range of doses (10−8 M to 10−5 M) apart from a small reduction (−20%) for an intermediate concentration (10−7 M) . This effect was only evident for early first trimester testes (8–9 GW), as there was no effect at any other gestational time-point examined (<8 GW or >10 GW) , and at 8–10 GW (10−4 M to 10−5 M); however, no significant difference in AMH was found after 7 days of exposure of host mice carrying second trimester human fetal testis xenografts  testis, an overall dose response reduction was demonstrated (Insl3 production was not affected in early first trimester (8–10 GW) human fetal testis following In-vitro culture and exposure of first trimester human fetal testis to ibuprofen for 7 days resulted in a reduction in gonocyte number (−22%). However, there were no significant changes to germ cell number following exposure of second trimester xenografted testis tissue (−4 to 10−8 M) , but not in late first trimester (10–12 GW) testes  to aspirin for 3 days did not affect Insl3 production  human fetal testis tissue  to indomethacin (10−4 M) for 72 h, reduced testosterone (−20%), whereas exposure at lower concentrations (10−5 M to 10−8 M) had no effect (−5 M) increased testosterone production (+20%), when first trimester (8–12 GW) testes were exposed in-vitro for a similar duration  to indomethacin (10−5 M) for 2 days did not affect Insl3 production  for 2 days did not affect germ cell number in first trimester (8–12 GW) human fetal testis cultures  is a synthetic estrogen that was used clinically to prevent spontaneous miscarriage and pre-term labor from the 1940 s until the early 1970 s . DES wasin-vitro culture of rat and mouse fetal testis, have reported a reduction in testosterone production following exposure to DES  for 3 days did not alter testosterone production  for 35 days resulted in no significant difference in testosterone production by second trimester (15–19 GW) testis tissue. Interestingly, host mouse seminal vesicles were significantly increased in weight, which was indicative of increased testosterone production from the xenografted tissue over the entire grafting period .Metformin is a biguanide used as an insulin sensitizer in the treatment of Type 2 Diabetes and obesity. Metformin may also be prescribed during pregnancy in individuals with pre-existing diabetes or those that have developed gestational diabetes during their pregnancy (in-vitro or in-vivo exposure. Exposure of pregnant mice to metformin (300 mg/gk/d) from embryonic day (e) 0.5 resulted in a significant reduction in testosterone at e16.5  resulted in a significant decrease in testosterone production from the testis. Importantly, the lowest concentration (5×10−5M) reflects the serum levels measured in humans following a therapeutic dose of metformin  and abiraterone are drugs known to inhibit key enzymes of the steroidogenic pathway including P450scc  and CYP17A1 & 3βHSD (abiraterone). As a result, exposure of the fetal testis to these agents could be predicted to affect testosterone production and potentially to result in the development of male reproductive disorders.Exposure to ‘azole’ antifungals that interfere with steroidogenesis may be relevant to pregnancy, given that they are frequently prescribed for the treatment of vaginal candidiasis. No association between maternal use of antifungals and hypospadias has been reported in two studies  in testosterone following for 96 h . SimilarAbiraterone (an anti-androgen used in prostate cancer) has also been shown to result in a reduction in testosterone production (−80%) in second trimester human fetal testis xenografts (grafted for 14 days), whilst no effect on germ cell number was demonstrated . Indeed,in-vitro system , albeit there appeared to be a modest decrease in testosterone production for caffeine only at the lowest concentration Table .Alcohol exposure during pregnancy is known to impact on fetal development, and its mechanism of action could occur centrally or in the placenta. Fetal alcohol syndrome is well described and can include neurodevelopmental disorders, facial dysmorphism and growth abnormality .An epidemiological study involving a Danish–Finnish cohort (~2500 boys), demonstrated an association between maternal alcohol consumption and increased risk of cryptorchidism in sons (−8 M to 10−5 M for 72 h) using in-vitro culture resulted in a significant increase in testosterone production across a wide dose range  in exposed- compared to unexposed-men , indicating that activation of the AHR is the likely mechanism for the effects of DMBA-DHD on germ cells  has been investigated in a recent study involving As expected, each mixture resulted in a reduction in testosterone production. Importantly, the authors compared the individual dose–response results to those predicted by the additive effect of the individual agents. There was a high correlation between predicted and actual response for each of the four mixtures indicating that these agents acted in an additive manner  approaches. However, this review has described several exposures for which the studies utilizing human tissues have demonstrated important differences to those found in animals. This may relate to differences in study design, dose administered or exposure regimen; however, it has also been shown that many of these differences appear to result from fundamental species differences in the effect of specific agents at human-relevant levels of exposure. Whilst epidemiological studies can to some extent bridge the gap between effects demonstrated in animal studies and human-relevance, such studies cannot demonstrate direct causation or elicit underlying mechanisms for effects; such studies are also prone to confounding. This highlights the importance of experimental models using human fetal tissues in determining the potential impact of in-utero environmental and pharmaceutical exposures in humans.Whilst a relatively large body of animal data exists for determining the impacts of in-utero exposures on male reproductive development will continue to rely on interpretation of a combination of animal studies, epidemiology and experimental studies utilizing human tissues. Conducting co-ordinated studies that combine these methods represents an important approach. This may include combining experimental studies using in-vitro and in-vivo approaches . Furthermore, for pharmaceuticals with relatively short half-lives, which are taken intermittently and do not accumulate in the body, obtaining accurate and detailed records of exposure during pregnancy is essential. This information is extremely difficult to obtain retrospectively and is prone to recall bias making prospective studies crucial for such exposures.Epidemiological studies may be enhanced by developing large-scale prospective cohort studies. This is of particular importance to ensure that the timing of measurement of exposure coincides with the expected mechanism of effect (e.g. in-utero exposure(s) on male testicular development and reproduction is also likely to involve refinement of existing experimental approaches. Whilst recent development of in-vitro and xenograft approaches has allowed direct testing of environmental chemicals and pharmaceuticals on the human fetal testis, these models may be limited by tissue supply and heterogeneity between individuals. The recent development of organoids for a variety of organs and tissues may prove critical for future studies to test the effects of exposures in organoids generated from human testicular tissues (in-silico (The future for determining effects of  tissues . ComputaThe scientific and public interest in the effects of chemical exposures in humans is likely to continue to increase. Regulation of these agents will increasingly rely on models that can provide direct human-relevant data. Therefore, we propose that assessment of the experimental evidence from studies using human fetal tissues should be an integral part of informing regulatory policies in relation to the effects of environmental and pharmaceutical exposures on male reproductive development.HRU-18-0051-R1-SuppTables_dmz004Click here for additional data file.


To describe patients with inherited and acquired complement deficiency who developed invasive meningococcal disease (IMD) in England over the last decade.Public Health England conducts enhanced surveillance of IMD in England. We retrospectively identified patients with complement deficiency who developed IMD in England during 2008–2017 and retrieved information on their clinical presentation, vaccination status, medication history, recurrence of infection and outcomes, as well as characteristics of the infecting meningococcal strain.A total of 16 patients with 20 IMD episodes were identified, including four with two episodes. Six patients had inherited complement deficiencies, two had immune-mediated conditions associated with complement deficiency (glomerulonephritis and vasculitis), and eight others were on Eculizumab therapy, five for paroxysmal nocturnal haemoglobinuria and three for atypical haemolytic uraemic syndrome. Cultures were available for 7 of 11 episodes among those with inherited complement deficiencies/immune-mediated conditions and the predominant capsular group was Y (7/11), followed by B (3/11) and non-groupable (1/11) strains. Among patients receiving Eculizumab therapy, 3 of the 9 episodes were due to group B (3/9), three others were NG but genotypically group B, and one case each of groups E, W and Y.In England, complement deficiency is rare among IMD cases and includes inherited disorders of the late complement pathway, immune-mediated disorders associated with low complement levels and patients on Eculizumab therapy. IMD due to capsular group Y predominates in patient with inherited complement deficiency, whilst those on Eculizumab therapy develop IMD due to more diverse capsular groups including non-encapsulated strains. Neisseria meningitidis (the meningococcus) remains a leading cause of bacterial meningitis and septicaemia worldwide, despite continued advances in the understanding of the pathogenesis of infection and development of new vaccines against this devastating infection. The meningococcus is commonly carried in the human nasopharynx, especially in adolescents and young adults [g adults , 2. In rg adults  have ideg adults . Defectsg adults . Individg adults .In addition to inherited deficiencies of the terminal complement pathway, a number of medical conditions and treatments can lead to acquired or secondary complement deficiency. In particular, Eculizumab  is a humanised monoclonal antibody that is a terminal complement pathway inhibitor used to treat paroxysmal nocturnal haemoglobinuria (PNH)  and atypCharacterising cases of IMD in individuals with complement deficiencies is fundamental to understanding disease risk in this highly vulnerable population and developing evidence-based guidance to both prevent and rapidly treat this potentially fatal condition. Public Health England (PHE) conducts enhanced national surveillance of IMD in England and routinely follows-up all cases confirmed by its national Meningococcal Reference Unit (MRU). Here we describe age distribution, clinical presentation, risk of recurrence, meningococcal typing and outcome of IMD in individuals with inherited or acquired complement deficiency diagnosed in England over a ten-year period.Neisseria meningitidis identified by culture or PCR from a sterile site. Confirmed cases are routinely followed up through postal questionnaires sent to their general practitioners for information on vaccination status, underlying co-morbidities, clinical presentation and outcomes of infection. For this study, IMD cases in patients with inherited or acquired complement deficiency diagnosed in England during 2008–2017 were included. Culture and non-culture samples submitted to the PHE MRU were characterised as reported previously [In England, National Health Service (NHS) hospital laboratories routinely submit invasive meningococcal isolates to the PHE MRU for confirmation, grouping and additional characterisation . The MRUeviously –16.A total of 16 complement-deficient patients with 20 episodes of IMD were identified during the surveillance period  which was first diagnosed in 2010 when the patient developed infective endocarditis needing mitral valve repair, ischaemic cerebral events, lower limb amputation and endophthalmitis resulting in complete loss of vision in one eye. This patient developed IMD 3 years later whilst on immunosuppressive therapy for the underlying condition. The second case with an immune-mediated condition affecting complement developed IMD in 2012 and was diagnosed with membranoproliferative glomerulonephritis associated with persistently low complement factors C3 and C4 four months later.Eight other patients were on Eculizumab therapy, five for paroxysmal nocturnal haemoglobinuria (PNH) and three for atypical haemolytic uraemic syndrome (aHUS). The age range of patients on Eculizumab therapy was 20 to 40 years with a median age of 22 years. Overall, cultures were available for 7 of 11 episodes in patients with inherited complement deficiencies or immune-mediated conditions, and 6 of 9 episodes in patients receiving Eculizumab therapy despite national recommendations for daily antibiotic chemoprophylaxis in this vulnerable group. The rest of IMD episodes were confirmed by PCR only.ctrA positive but not positive for capsular groups A, B, C, W or Y), suggesting a truly non-groupable strain or a rare capsular group. For those on Eculizumab therapy, six of the nine isolates were due to either capsular group B (n = 3) or non-groupable (interruption of ctrA gene) but genotypically capsular group B (NG(B); n = 3). Three other episodes were due to capsular groups W, Y and E. Capsular group Y strains was mainly associated with PorA P1.5–1,10–1. One isolate was penicillin-resistant - a non-groupable strain that was genotypically characterised as group B [The predominant capsular group for the inherited complement deficiencies or immune-mediated conditions was capsular group Y (7/11), followed by capsular group B (3/11); one strain that was non-groupable by PCR  at the time and only received the MenACWY conjugate vaccine after the second episode of IMD. Case 3 with complement C8 deficiency had two episodes of IMD at 20 and 42 years of age due to capsular group Y and B, respectively; this patient was prescribed prophylactic amoxycillin twice daily following the second episode. Case 5 was diagnosed with complement C6 deficiency after the second IMD episode. This case developed IMD due to capsular group B at 4 years of age and then with capsular group Y at 16 years; the patient had received the MenACWY conjugate vaccine as part of the national UK adolescent programme 2 years prior to the second episode of IMD. Case 16 with aHUS commenced Eculizumab therapy aged 20 years and presented with IMD at 20 and 22 years of age after been appropriately vaccinated with a single dose of MenACWY conjugate vaccine and two doses of the meningococcal group B vaccine, 4CMenB. This patient was reported to be intermittently non-compliant with antibiotics. Both IMD episodes were confirmed by PCR only; the infecting strains were both PCR negative for the We identified 16 individuals with invasive meningococcal disease who had confirmed primary or acquired complement deficiency in England over a decade. Most cases developed IMD during adolescence and early adulthood, including four who developed recurrent IMD; three of the four cases were diagnosed with an underlying primary complement deficiency after the second IMD episode. In addition to the primary complement deficiencies, we identified two groups with acquired complement deficiency, those with immune disorders leading to secondary complement deficiency and iatrogenic cases in patients receiving Eculizumab, a humanised monoclonal complement C5 inhibitor.The complement system is an important component of the innate immune defence against invading bacteria including meningococci; complement activation on cell surfaces may be initiated through any of the three pathways - the classical, lectin, or alternative pathways. The terminal complement pathway comprises five proteins (C5-C9) that combine together to form a lethal pore-like membrane attack complex (MAC), which disrupts the bacterial cell membrane and forms transmembrane channels, leading to bacterial lysis . ComplemIn our cohort, we observed an over-representation of IMD due to capsular group Y in those with inherited complement deficiencies  compared to those receiving Eculizumab therapy ; the risk of group Y IMD in the latter group is similar to the rest of the population - in England, IMD due to group Y was responsible for 10.9% of all cases during 2011–15 . During Another significant difference between the inherited complement deficiencies and the patients on Eculizumab therapy was that all but one strain  causing IMD in patients with inherited complement deficiencies were encapsulated, as were both the strains causing IMD in patients with immune-mediated conditions associated with complement deficiency (both group Y). In contrast, four of the nine IMD episodes in patients on Eculizumab therapy were either NG or group E; these strains are less virulent and usually only associated with carriage.Our findings support the recent French report of 56 cases with terminal complement pathway deficiency who developed 61 episodes IMD between 1980 and 2015 [A new and significant finding in our cohort was that there were more patients with IMD who were on Eculizumab treatment than those with primary terminal complement deficiency, including six treated for PNH and three for aHUS. An unwanted complication of complement inhibition, however, is an increased risk of infection with encapsulated bacteria, especially IMD. Unfortunately, just like patients with primary terminal complement deficiency, this group remains at increased risk of IMD even after appropriate immunisation because they are unable to mediate meningococcal lysis by the membrane attack complex. This finding emphasises the importance of penicillin chemoprophylaxis to prevent IMD and other serious bacterial infections in high-risk patients. Whilst there are no published guidelines on the management of individuals with inherited complement deficiencies or immune-mediated conditions associated with hypocomplementaemia, antibiotic chemoprophylaxis is routinely recommended for those with asplenia and those receiving Eculizumab. We have, however, recently reported a case of penicillin-resistant MenB disease in a vaccinated young adult on penicillin prophylaxis, highlighting the need to raise awareness of IMD risk among patients and healthcare professionals, as well as the need to seek early medical attention and for early investigation and treatment in unwell patients receiving Eculizumab, who usually present with non-specific symptoms and signs of infection. Although none of the patients in our cohort died of IMD, there have been reports of fatalities due to NG strains in patients receiving Eculizumab .In addition to the known risk factors for IMD, we found other rare conditions associated with complement deficiencies, such as hypocomplementaemic urticarial vasculitis (anti-C1q vasculitis), which is associated with glomerulonephritis  as well IMD in patients with complement deficiency is generally associated with mild disease, possibly due to a less intense inflammatory response in the absence of an intact complement pathway. Fatal cases have, however, been reported, including IMD cases due to NG meningococci, which generally only attack vulnerable hosts . In addiAcquired and inherited deficiencies of the terminal complement pathway are rare, even in patients with IMD. Most cases of IMD in patients with inherited complement deficiency occur during adolescence. Testing for complement deficiency after a single episode of IMD is not routinely recommended because the vast majority of patients do not have any underlying co-morbidities. Healthcare professionals should, however, consider the possibility of inherited complement deficiency in any patient with recurrent IMD or in those who develop IMD due to unusual capsular groups such as Y, E or NG meningococci, especially in previously healthy adolescents. The management of such patients is challenging because of the limited protection offered by the current vaccines. In addition to vaccination, antibiotic chemoprophylaxis should be strongly considered but rare cases of penicillin-resistant group B meningococcal disease highlight the need to raise awareness of IMD risk among patients and healthcare professionals, including use of information cards to be carried by patients and their care givers, as well as the need to seek early medical attention and for early investigation and treatment in unwell patients.