Low-visibility events (LVEs) are severe weather phenomena that are closely linked with anthropogenic pollution, which negatively affects traffic, air quality, human health, and the environment. This study conducted a two-month (from October to December 2019) continuous measurement campaign on Chongming Island in Shanghai to characterize the LVEs transition and its drivers. The LVEs accounted for 38% of the time during the campaign, of which mist accounted for 14%, fog–haze for 13%, haze for 6%, and fog for 5%. The fog and mist mainly occurred from midnight to early morning, while haze mostly occurred during the daytime. Different LVEs were interdependent and transitioned from one to another. Fog generally turned into haze after sunrise, while haze turned into fog after sunset. Their formation and evolution were caused by the combined impacts of meteorological conditions and aerosol particles. It was found that temperature difference was the dominant meteorological factor driving the evolution of LVEs. Within the short term, cooling led to a greater increase in relative humidity than humidification. Radiative cooling during the night promoted the formation of fog and mist. During fog and mist events, cloud condensation nuclei (CCN) were mainly internally mixed due to the impact of fog droplet removal and aqueous/heterogeneous aerosol reactions occurring under high humidity. Increased CCN concentration appeared to increase the fog droplet number and liquid water content in fog events. Overall, conditions of high humidity and high particle loading were conducive to LVEs, whereas conditions of sufficient water vapor at a low particle level and sufficient particles at a low humidity level also caused LVEs. This study provided insights into LVEs classification, evolution scheme, and aerosol roles from a micro point of view. The findings could be useful for improving forecasts of local radiative fog and other LVEs. Low visibility events (LVEs) associated with air pollution have occurred frequently and widely in China in recent decades, posing adverse effects on traffic and human health ,2. LVEs LVEs are attributed to the synergistic impacts of anthropogenic emissions and meteorological conditions in and around urban regions ,6,7,8. USoluble aerosols such as sulfate, nitrate, and sea salt, and coarse aerosols such as dust, which can act as CCN, have important contributions to cloud/fog formation and precipitation ,27,28,29In order to explore the evolution of LVEs, we selected Chongming Island for a field observation site, located in the marginal intersection of the ocean and the Yangtze River Delta (YRD) region, which was readily subjected to abundant anthropogenic pollutants and sufficient water vapor. The previous method to classify low visibility weather using average daily visibility and relative humidity made it difficult to distinguish LVEs at the time scale of less than one day and capture weak LVEs and LVEs transitions such as the coexistence of haze–fog events ,38,39. TAutumn and winter were affected by particulate matter pollution and stable weather conditions, which were the seasons having a high incidence of LVEs and were often accompanied by the mutual transition of LVEs. The field campaign was conducted from October through December (122 days) in 2019 at the Meteorological Observation Station on Chongming Island of Shanghai  at six discrete supersaturation (ss) levels (0.1–1.0%). The 500 cmlsewhere ,42.3/min flow . As shown in 3/min and was then split into two streams to enter the CPC and CCN counters.One set of Scanning Mobility Particle Sizer (SMPS) was employed to measure size-resolved particle number concentrations within 10–350 nm, consisting of Long Differential Mobility Analyzer  and Condensation Particle Counter with 300 cm3 min−1. Fog droplet number concentration (Nd), liquid water content (LWC), and effective diameter (ED) were computed using the measured droplet spectrum.The Fog Monitor  was used to measure in situ the real-time size distributions of 2–50 μm fog droplets with a sampling rate of 1 mWith the development of meteorological modernization, manual observations have been replaced with automatic acquisition instruments. Atmospheric visibility (VIS) has been gradually shifted from manual to machine observations since 2013 in China. The meteorological optical range (MOR) observed by the automatic Forward Scattering Visibility Meter, is defined as the path length in atmosphere required to reduce the luminous flux in a collimated beam, which is emitted from an incandescent lamp at a color temperature of 2700 K, to 5 percent of its original value ,44. Howecorr  to condensation nuclei number concentration (NCN). Aerosol CCN activity spectrum is fitted with a cumulative Gaussian  distribution function using a non-linear least-squares fitting routine with three parameters  [Aerosol activated ratio (AR) is defined as the ratio of CCN number concentration fNCl mixing . The hetc (σ/Dc) .kS is saturation ratio, D is droplet diameter, pD is the diameter of the dry aerosol particle. κ is the hygroscopicity parameter of aerosol particles, which is calculated from the critical saturation ratio (cS) and critical diameter (cD) from the following equation:ρw is the density of pure water (about 997.04 kg m−3), Mw is the molecular weight of water (0.018 kg mol−1), σw is the surface tension of solution–air interface, which is assumed to be the value of pure water (0.0728 N m−1), R is the universal gas constant (8.314 J mol−1K−1), T is the temperature in Kelvin (298.15 K), and Dc is the critical diameter (m).The parameter kappa (κ) has been generally used to characterize the hygroscopicity of aerosol particles . The κ-KCN was up to 15,259 cm−3, which was consistent with early observations of 15,000 cm−3 during haze episodes in Shanghai [−3 during winter in Guangzhou [CCN increased along with the SS and was related well to the NCN.A series of LVEs occurred on Chongming Island in October and December of 2019. Mist occurred in 65 days, and fog occurred in 26 days of 122 days. In addition, fog–haze occurred in 62 days, and haze occurred in 40 days. The frequency of LVEs classified on a 1 h time scale was 38.08% during the observation period, which was dominated by mist (14.22%), followed by fog–haze (13.22%), haze (5.75), and fog (4.89)%. Besides LVEs, rain accounted for 7.97% and clean events accounted for 53.94%. Shanghai  (Leng etuangzhou  (Duan etd), 12 h temperature gradient (T-T12), and LTS during LVEs. T-Td requirements for fog and mist formation were the most restrictive. A bimodal distribution of fog and mist occurring between −0.2 and 0.2 °C was observed. The first peak, in which fog and mist occur at 65% and 56%, stretched between −1.0 and −0.6 °C, while the second peak was located between −1.0 and −0.6 °C. Fog–haze T-Td presented a unimodal distribution within 0.6–1.0 °C, with a peak frequency of 58%. The T-Td frequency spectrum of haze events varied between 1.0 and 10.6 °C. T-Td of 0.2 °C could be considered as the threshold for separating fog–haze and fog/mist events, whereas T-Td of 2.2 °C could be used for separating haze and fog–haze events.12. T-T12 showed distinct features according to weather types as well. About 94% of the fog and mist events had negative T-T12 values, mostly less than −8 °C, providing evidence of the impact of terrestrial energy release on the formation of radiation fogs at night [12 of the fog–haze events were mostly between −6 and −4 °C, with a maximum frequency of 21%, indicating that fog–haze was partly influenced by radiation cooling. When haze occurs, it usually occurred during the daytime, and its corresponding T-T12 was almost always positive (0–8 °C). When fog occurred, frequencies tended to concentrate at LTS larger than 9 K (79%), showing higher stability than other LVEs. LTS for haze events was relatively small, with an average of 6.24 K, even less than for clean events (7.63 K). The stable atmosphere restrained the vertical mixing of particulate matter and water vapor, conduced to gather particles and droplets within the boundary layer, and then formed haze or fog events and maintained them. The radiation cooling of fog and mist can be quantified using T-Tat night ,58. The Previous studies have also revealed that increased atmospheric stability allowed particles to accumulate near the surface and caused air pollution in the North China Plain (NCP) and the YRD ,59. In aCN and NCCN) and CCN activation curve under various atmospheric conditions. The NCN and NCCN of the fog–haze event were the highest among all types of LVEs, followed by haze, mist, and fog, which suggested the removal effect of the fog droplets on particulate matter [pD), while the activation curves of haze and clean period showed relatively flat patterns, inferring stronger inhomogeneity of aerosol population and higher external mixing degree in the latter [e matter . The pare latter . The actσ/cD) with particle critical activation diameter (cD). As for aerosol hygroscopicity, the dual effects of particle size and water vapor on aerosol activation were described in classic Köhler theory [σ/cD), the internal mixing particles  with ignorable heterogeneity (zero) were preferentially activated at low SS levels., The particle heterogeneity increased at high SS levels, indicating that favorable water vapor conditions allowed external mixing particles  with different compositions emitted from various sources to grow and even enhance CCN loadings. As shown in σ/Dc values usually occurred during fog and mist events and hinted at intensive internal mixing of particles compared to other events. This result was slightly different from the previous study in LinAn [r theory , that war theory ,65. Ovadr theory  also poiin LinAn , likely in LinAn ,67.Several fog weather processes were captured by FM-120 during the field campaign. The temporal variations of the fog-droplet size distributions on 1 and 2 November are depicted in Known as a general rule of cloud physics, given almost invariant or limited LWC in clouds, the entrained aerosols can increase the concentration of droplets ,69,70. LCN increased by 5.96% when a clean event turned into the haze, while NCCN increased by 9.63% when a clean event turned into the fog–haze. In addition, it was found that the CCN reduction (−1454 cm−3) was more significant than that of CN (−1271 cm−3) when fog turns to haze, supporting the role of the fog droplets in the scavenging of CCN [T caused a greater change in relative humidity than Δq. Temperature decrease was more likely to cause RH to increase than a humidity increase, and temperature increase was also more likely to cause RH to decrease, even with a strong humidity increase. Usually, the rate of change of T exceeds that of q by an order of magnitude . The value presented here is based on the statistical analysis of this study. A larger number of field campaigns were needed to investigate whether this threshold was universal for regions and seasons beyond Shanghai. However, this conceptual model and threshold will be extremely valuable in understanding the formation, evolution, and transition of LVEs.Shanghai Chongming had been plagued by sporadic or continuous LVEs throughout the winter of 2019. A field campaign was conducted to investigate the LVEs from October to December of that year. The previous method to classify low visibility weather using average daily visibility and relative humidity made it difficult to distinguish LVEs at a time scale of less than one day and capture weak LVEs and LVEs transitions, such as the coexistence of haze–fog events. This study took measures to resolve LVEs on an hourly time scale and better understand the evolution of LVEs and their related driving factors from a microscopic viewpoint.The total frequency of hourly low-visibility events was as high as 38.08%. Different LVEs existed interdependently, among which fog–haze events with relatively high frequency (13.22%). LVEs exhibited significant diurnal variations, which could be explained by the variation in temperature, wind speed, low tropospheric stability, and aerosol concentration. Fog and mist events mostly occurred at night, especially from midnight to early morning, whereas haze events mostly occurred in the daytime. More than 35% of the haze events had wind speeds greater than 3 m/s in Chongming Island, in which the north wind contributed the most, indicating that haze in this region was mostly caused by long-distance transported aerosols from inland. For fog and mist events, the surface radiation cooling effect was significant at night. At the same time, the lower boundary layer height inhibited the vertical diffusion of water vapor at night, which accumulated near the ground and formed radiation fog.In terms of microphysical properties, as the aerosols hygroscopicity increased, the inhomogeneity of aerosols gradually decreased. However, the aerosols in fog and mist events showed relatively low hygroscopicity and a high degree of internal mixing due to the removal effect of high hygroscopicity particles by fog droplets and the heterogeneous or/and liquid-phase reactions of aerosols under high humidity, respectively. The number concentration, effective diameter, and liquid water content of the fog droplets were more remarkable in high CCN conditions than in low CCN levels.d dropped to 0.2 °C, and NCN reached 3000 cm−3, while when T was greater than 2.2 °C, NCN needed to rise twice (6000 cm−3) to form haze.The formation and evolution of LVEs were the results of the joint impact of anthropogenic pollution and meteorological conditions. High ambient humidity and particle concentrations were certified to operate LVEs; otherwise, higher water vapor at low particle level (or higher particle concentrations at low humidity level) was also a trigger mechanism. The temperature difference was the most critical meteorological driving factor in the evolution of LVEs. Cooling was more likely to improve the ambient saturation than humidification in the short term. Fog or mist was triggered when T-TQuestions surrounding how atmospheric physical and chemical processes can affect the formation and evolution of LVEs merit further exploration. In the future, we will estimate the contribution of individual driving factors to LVEs, and focus on the influence of aqueous reactions and the aerosol–droplet–meteorology interactions on the evolution of LVEs.


To characterize a suggestive prenatal imaging pattern of Aicardi syndrome using ultrasound and MR imaging.Based on a retrospective international series of Aicardi syndrome cases from tertiary centers encountered over a 20‐year period (2000–2020), we investigated the frequencies of the imaging features in order to characterize an imaging pattern highly suggestive of the diagnosis.Among 20 cases included, arachnoid cysts associated with a distortion of the interhemispheric fissure were constantly encountered associated with complete or partial agenesis of the corpus callosum . This triad in the presence of other CNS disorganization, such as polymicrogyria , heterotopias , ventriculomegaly , cerebral asymmetry ) and less frequently extra‐CNS anomaly  represent a highly suggestive pattern of Aicardi syndrome in a female patient.Despite absence of genetic test to confirm prenatal diagnosis of AS, this combination of CNS and extra‐CNS fetal findings allows delineation of a characteristic imaging pattern of AS, especially when facing dysgenesis of the corpus callosum. What is already known about this topic?Aicardi syndrome (AS) is a rare developmental encephalopathy, characterized by the classic triad of infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunaeAs the genetic etiology of AS is unknown and its classic triad cannot be extrapolated to prenatal diagnosis, to establish a prenatal imaging pattern is of major interestWhat does this study add?We confirmed and completed a prenatal imaging pattern highly suggestive of AS, based on the most frequent features encountered in a large series: arachnoid cysts associated with a distortion of the interhemispheric fissure, agenesis of the corpus callosum, cortical malformations and heterotopias, and less frequently extra‐CNS anomaly  In addition to the classic triad, major features have been described, mainly using cerebral MR imaging (MRI): cortical malformations (polymicrogyria), periventricular and subcortical heterotopias, arachnoid cysts around the third ventricle, choroid plexus cysts, and optic nerve/disc coloboma or hypoplasia. Additional features can also be suggestive of the diagnosis especially when the classic triad is incomplete: cerebral asymmetry, vertebral and rib malformations , microphthalmia, vascular malformations, split‐brain EEG , and facial dysmorphism including prominent premaxilla with upturned nasal tip and sparse lateral eyebrows.Diagnosis of AS based on the classic triad cannot be extrapolated to prenatal diagnosis as seizures and chorioretinal lacunae cannot reliably be diagnosed before birth. Indeed, AS can only be suspected if major or/and additional features are seen in a female fetus who has normal genetic testing results. Therefore, AS is rarely diagnosed prenatally and only a small number of cases have been reported.22.1This study is a retrospective analysis of cases of AS prenatally suspected after a dedicated prenatal imaging workup, including sonographic and MRI exams, performed in tertiary centers, over a 20‐year‐period (2000–2020) and pathologically/postnatally confirmed. Cases were collected from the following centers: Lausanne University Hospital , HFME , Lis Maternity Hospital , Diagnus , Hospital of Children Ricardo Gutiérrez , Medical University of Vienna and Medical University of Graz (Austria), Mount Sinai Hospital , Gaslini Children's Hospital , Texas Children's Hospital , Massachusetts General Hospital . The study was approved by the local Institutional Review Boards according to each country's requirements.2.2Criteria for inclusion in our study were the following: (1) Suspicion of AS after a prenatal evaluation in a tertiary center; (2) Prenatal and pathological/postnatal data and imaging available for review; (3) Pathological/post‐natal confirmation of AS based either on presence of the classic triad  or presence of distinctive chorioretinal lacunae associated with another anomaly of the classic triad plus at least two other major or additional features either at autopsy or during clinical work‐upCases were excluded from the analysis if (1) Etiological work‐up found another diagnosis; (2) Pathological or postnatal confirmation of AS diagnosis was not available; (3) Assessment or imaging quality do not correspond to guidelines for dedicated neurosonography and fetal MRI at the time of the examination , through a datasheet via the secure messaging system of Lausanne University Hospital. Each referent kept the source file allowing to link the identity of the patients of their center with the anonymized number used for the study.2.4Prenatal sonographic and MR iconography was reviewed  to identify features associated with AS. The quality of the examinations was evaluated based on ISUOG guidelines.MRIs were considered of “good” quality if a three‐plane examination of the fetal brain, eyeballs, optic nerves and retina was available in at least one T2 weighted‐contrast sequence . MRIs were considered of “sufficient” quality if a three‐plane examination of the fetal brain and eyeballs was available. MRI scans were considered to be of “insufficient” quality if three‐plane fetal brain and/or eyeball scans were not available.33.1Between 2000 and 2020, 28 cases of AS were suspected based on prenatal imaging from the 12 international centers involved in our study. Pathological/post‐natal confirmation of the diagnosis was available for 20 cases (71.4%). The eight unconfirmed cases included seven patients lost to follow‐up after prenatal diagnosis, and one case in which a final diagnosis of oculo‐cerebro‐cutaneous syndrome was established after birth.The 20 cases included had pathological/postnatal confirmation of the diagnosis based on the presence of the classic triad  or at least presence of chorioretinal lacunae associated with another anomaly of the classic triad and at least two other major or accessory features.Fourteen patients (70%) were primigravida whereas six were multigravida, of whom three (15%) had one or more miscarriages prior to this pregnancy, without any personal or familial history of congenital anomalies. One patient (Case 6) had a periconceptional cytomegalovirus seroconversion for which congenital infection was ruled out by negative PCR in amniotic fluid and neonatal urine. Apart from one case with nuchal translucency >95th percentile at 12 weeks of gestation (w), no abnormality was detected by first trimester ultrasound or serum markers .3.2All included patients were referred for suspected corpus callosum anomalies and/or cerebral cysts and/or fetal ventriculomegaly in the second or third trimester. The median gestational age at the time of the referral ultrasound was 23w (18 to 37w). Fifteen patients underwent fetal brain MRI, performed at a median gestational age of 31w (22 to 36w). Description of MR protocols, as well as data on prenatal sonographic and MR examination quality, are presented in Table 3.33.3.1Extra‐axial cysts were present in all cases Table  and 2 loChoroid plexus cysts were described in three cases . In one case, the choroidal plexus cyst was unusually large and associated with a solid component demonstrating high Doppler signal, suggestive of choroid plexus papilloma.3.3.2Distortion of the interhemispheric fissure was demonstrated in all cases (20/20), giving an asymmetrical aspect of the frontal lobes in 14 cases (70%).3.3.3ACC was diagnosed in all cases, except one , since in the three older cases the imaging quality precluded detection of such anomalies. Diffuse versus focal polymicrogyria was shown in three  and 13 cases respectively, predominantly in frontal location, with unilateral involvement in six cases.Neuronal migration abnormalities were observed in 15 cases . Heterotopia and nodules were located in the periventricular , subependymal , and subcortical areas . Heterogeneous aspect of the basal ganglia was noted in three cases , consistent with heterotopias. Schizencephaly was suspected in four cases (20%). In Cases 2 and 3, closed‐lip communication between pericerebral space and ventricles, bordered by polymicrogyria, was suggestive of a true schizencephaly reflecting an early ischemic lesion, whereas in Cases 14 and 17, the aspect was related to extreme thinning of the parenchymal mantle facing unilateral severe ventriculomegaly leading to near‐communication between the ventricular lumen and the subarachnoid space.In four cases , MRI diagnosed cortical abnormalities which were not described on ultrasound, and in three other cases  MRI confirmed suspected heterotopias sonographically described as “hyperechoic foci.”3.3.5Unilateral versus bilateral ventriculomegaly were observed in nine  and five  cases, respectively . These ventriculomegalies were severe in eight cases . On ultrasound, a thickened, hyperechoic and heterogeneous ventricular wall was described in several cases , suggesting periventricular migration disorders. On MRI, occipital and frontal periventricular irregular hyperintensities and nodules were described in these cases, also suggesting heterotopias.3.3.6Posterior fossa anomalies were found in seven cases (35%). Cases 1, 5, 14, and 18 presented a cystic malformation of the posterior fossa: arachnoid cyst in three cases and a Blake's pouch cyst in the last one. An enlarged cisterna magna was found in case 16. Cases 5, 11, 14, and 15 showed asymmetry of the cerebellar hemispheres, with abnormal foliation of the inferior border of the left cerebellar hemisphere in Case 5, and unilateral hypoplasia of one hemisphere in Cases 14 and 15.3.3.7Ocular anomalies were identified mainly in the most recent cases displaying high prenatal imaging quality and providing detailed analysis of the globes, optic nerves and retinas (11 cases).Of these cases, nine had abnormalities . Microphthalmia was found in seven cases , unilateral in six and bilateral in one. Retinal or optic nerve colobomas were found in seven cases , described as bilateral/unilateral in one and six cases, respectively  had in utero repeated spastic movements, suggestive of seizures. Vertebral anomalies were identified in four cases (20%) . These anomalies were scoliosis with thoracic  and lumbar (Case 9) hemi‐vertebrae Figure .Of the examinations providing 3D imaging of the fetal face, dysmorphism consistent with the one described in ASs was identified in four cases 4/10, 40%)   strongly suggested a choroid plexus papilloma, a tumor described in AS.3.4Pathological and postnatal features are summarized in Table Six pregnancies 30%) were terminated on parental request because of poor expected post‐natal neurodevelopmental outcome. Both brain parenchyma and retinal fetal examination confirmed the diagnosis in all cases. Indeed, pathological examination confirmed chorioretinal lacunae in all cases  and four  cases respectively. Microphthalmia was found in five cases  . Retinal or optic nerve colobomas were found in seven cases (50%) , described as bilateral and unilateral in four  and three  cases respectively. Discrepancy with prenatal examinations suggestive of none anomalies or unilateral ocular anomaly that turned out to be present or bilateral postnatally, was found in three cases.3.4.2In the seven cases in whom data were available, EEG was abnormal. Cases 2, 14, and 16 showed abundant multifocal epileptiform discharges with asymmetric background activity and epileptic spasms on neonatal examination. Case 2 had focal epilepsy with impaired awareness and unstructured background activity. For the three remaining cases, no precise data regarding EEGs exam could be collected.3.4.3Postnatal MRI was performed for all newborns and confirmed prenatal imaging in all cases, providing additional information in six cases .3.4.4For all live births, clinical data could be collected at different stages of life (from one month to 12 years). Epilepsy or spasms were present in 13 of them (93%), described as drug‐resistant in four cases. In addition, 10 children (66%) who were examined during childhood, had severe neurodevelopmental delay . After the first year of life, the majority of children had multiple disabilities . Two presented with digestive complications (14%).3.4.5Vertebral anomalies were identified in six cases (43%)  including scoliosis with thoracic and lumbar hemivertebrae.3.4.6Karyotyping and CGH array, performed in 19 and five cases respectively, identified no chromosome number abnormalities nor abnormal copy number variations. Exome sequencing, performed in the five most recent cases, found no known pathogenic variants.4The imaging findings reported in our series are in concordance with those previously described in prenatal reports and post‐natal series of AS. Hopkins et al. reported cerebral MRI findings of 23 patients with AS. All had corpus callosum agenesis , polymicrogyria  and heterotopias . The majority of patients (95%) had also intra‐cranial cysts, mainly interhemispheric, and fossa posterior anomalies.In our series, distinctive chorioretinal lacunae were confirmed in all cases but we may have overestimated the frequency of this anomaly, as it was a nonexclusion criterion. These lacunae were present in 88%–100% of cases in other series.Dysmorphic features may be difficult to identify on prenatal imaging. We identified prenatal dysmorphic features in 40% of cases although a distinctive dysmorphism after birth was present in all cases. Indeed, the sagittal view of the profile can only identify the prominent maxilla described in AS. The use of 3D imaging may lead to a better identification of the other dysmorphic features: upturned nasal tip and upper lip, micrognathia and sparse lateral eyebrows (Figure In keeping with our main objective, based on the most frequent prenatal imaging findings encountered in our series, the most suggestive prenatal imaging pattern is represented by a triad, which includes one or more extra‐axial cysts associated with distortion of the interhemispheric fissure and partial or complete ACC in a female fetus. This diagnostic triad can be reinforced by major CNS organization anomalies, such as cortical/migration anomalies, or by ocular anomalies and less frequently vertebral anomalies. Regarding the ophthalmological anomalies, one should note that the pathognomonic chorioretinal lacunae, found in all cases of our series on post‐natal fundoscopy, could not be diagnosed on prenatal imaging. Therefore, in the absence of both genetic testing and prenatal access to chorioretinal lacunae, our prenatal imaging pattern based on the previously described features, is of major interest to highly suggest the diagnosis of AS in the female fetus, especially when dealing with prenatal counselling of ACC due to the poor cognitive and ophthalmological prognosis of this condition.Finally, we should underline the strength and the main limitations of our study. Indeed, this series represent the largest one of prenatally suspected cases of AS, based on the recruitment of international referral centers, which were expected to provide high quality examinations. The main limitation is the study period of 20 years, which was required to collect a large number of cases due to the rarity of this condition, and which obviously introduced some bias especially related to the evolution of imaging and genetic testing over time. Indeed, cases prior to 2010 were mainly studied by karyotype and, those between 2010 and 2017, by array‐CGH, due to difficult access to WES. Array‐CGH can detect deletions and duplications associated with other syndromes that have some features which mimic those of AS,5Finally, despite the lack of a genetic test to enable a prenatal diagnosis of AS, prenatal imaging can identify a combination of CNS and extra‐CNS features which form a characteristic imaging pattern of this syndrome, especially when facing intra‐cranial cyst associated with distortion of the inter‐hemispheric fissure and callosal dysgenesis.www.icmje.org/coi_disclosure.pdf and have no conflicts of interest to declare.All authors have completed the ICMJE disclosure form at Supporting Information S1Click here for additional data file.Supporting Information S2Click here for additional data file.


Infantile nystagmus syndrome (INS) causes altered visual development and can be associated with abnormal retinal structure, to which vascular development of the retina is closely related. Abnormal retinal vasculature has previously been noted in albinism but not idiopathic infantile nystagmus. We compared the number and diameter of retinal vessels in participants with albinism (PWA) and idiopathic infantile nystagmus (PWIIN) with controls.Fundus photography data from 24 PWA, 10 PWIIN, and 34 controls was analyzed using Automated Retinal Image Analyzer (ARIA) software on a field of analysis centered on the optic disc, the annulus of which extended between 4.2 mm and 8.4 mm in diameter.P < 0.001), and venous branches were reduced in both PWA  and PWIIN . PWA demonstrated 7% thinner “primary” (before branching) arteries , and 13% thicker (after branching) “secondary” veins .Compared with controls, the mean number of arterial branches was reduced by 24% in PWA (15.5 vs. 20.3, PWA and PWIIN demonstrated reduced retinal vessel counts and arterial diameters compared with controls. These changes in the superficial retinal vascular network may be secondary to underdevelopment of the neuronal network, which guides vascular development and is also known to be disrupted in INS. FRMD7 mutations,Infantile nystagmus syndrome (INS) is defined as rhythmic, involuntary oscillations of the eyes,,Albinism refers to a group of heritable conditions characterized by hypopigmentation of the skin, hair, and eyes, for whom numerous genetic mutations have been identified. These include oculocutaneous forms (OCA1-8), purely ocular forms (OA1), or as part of syndromic conditions, such as Hermansky Pudlak syndrome (HPS1-11).TYR gene cause impairment of the tyrosinase function of converting tyrosine to L-DOPA.Pigmentary deficits are caused by mutations affecting key enzymes across the melanin synthesis pathway. The mechanisms underlying pigmentation are complex. A sophisticated control mechanism exists with tyrosinase activity being modulated using the internal melanosome pH, which is in turn is determined by melanosome membrane potential controlled through a combination of ion channels.,Many of these visual deficits stem back to delayed neurogenesis in the retina, possibly related to reduced expression of L-DOPA during visual development.,There is strong evidence that the development of neural and vascular systems share common genetic and signaling pathways,In this study, we used retinal vessel analysis software to assess and compare the number and diameter of retinal vessels, in an annulus around the optic disc, in participants with albinism (PWA) and idiopathic infantile nystagmus (PWIIN) with controls. The data were also correlated with severity of FH.This prospective observational study included the data of 24 PWA  and 10 PWIIN . Participants were recruited from the neuro-ophthalmology clinic at Leicester Royal Infirmary, UK. Approval to conduct the study was gained from the local ethics committee and informed consent from all participants was obtained with adherence to the tenets of the Declaration of Helsinki.Diagnosis of PWA was confirmed by the presence of phenotypic features fulfilling criteria presented by Kruijt et al. (2018),All participants with ocular comorbidities and high refractive errors , systemic comorbidities associated with retinal vascular diseases, such as systemic hypertension, chronic renal disease, diabetes, blood hyperviscosity, and anemia were excluded from the study.Complete ophthalmic examination was performed on all participants, including best-corrected visual acuity, refraction, orthoptic assessments, slit lamp examination, fundus and OCT imaging, and eye movement recordings for those with nystagmus. OCT imaging  was used by one investigator (author S.T.) to grade the degree of FH using the standardized foveal hypoplasia scale developed by Thomas et al. (2011).NM/FA  fundus camera. A full field quadratic and feathered montage was created from the images using i2k RETINA software (standard version 2.4) from DualAlign. The eye with the higher quality fundus image was selected for the study.Using 1% tropicamide eye drops for mydriasis, 45 degree color fundus pictures of both eyes were taken using a Zeiss VISUCAMVessel detection and measurements were performed using the standardized settings of Automated Retinal Image Analyzer (ARIA), an open source, MATLAB  based program.The lack of pigmentation in some PWAs increased the prominence of choroidal vasculature, particularly beyond the major vascular arcades see . This grThe vessels were manually classified by type  and quadrant . Vessels which could not be identified as arterial or venous were excluded (33% of participants averaging 1.9 excluded vessels each) from the analysis. For each of these categories, mean vessel quantity and diameter was then calculated separately for “primary” vessel, “secondary” vessel, and “tertiary” vessel branches, as well as their total, using a systematic ordering method; vessels emerging from the optic disc were classified as “primary,” and at branching points thereafter the thinner vessel branch was labeled a “secondary” vessel and the thicker branch would continue as the “primary” vessel. Tertiary vessels were derived from secondary vessels in the same manner see .n = 6 and grade 2 n = 6) and “severe” FH (grade 3 n = 2 and grade 4 n = 10) subgroups, and the analysis repeated in comparison to the control group.As foveal changes are more frequently observed in PWA, the effect of degree of FH on vascular parameters was explored in this group. The 24 participants of the PWA group were divided into “moderate” FH P values adjusted using Bonferroni correction. P ≤ 0.05 was considered statistically significant.SPSS software version 24.0  was used. Vascular parameters were normally distributed. ANOVAs were used to compare mean vessel quantity and mean vessel diameter among the PWA, PWIIN, and control groups. Significant differences were explored using a post hoc analysis with Because albinism is associated with retinal nerve fiber layer thinningP < 0.001 and veins = 12.9 compared to 18.2, P < 0.001, respectively; P < 0.001 and tertiary = 0.92 compared to 3.09, P < 0.001, respectively) and veins  in PWA as compared with controls, indicating reduced branching. Tertiary arteries and veins were only detected in 54% (13/24) and 58% (14/24) of PWA, respectively, which was less than in controls – 91% (31/34), Chi-square = P = 0.001) and 82% (28/34), P = 0.044, respectively. Numbers of PWIIN in whom tertiary arteries and veins could be detected was also lower – 60% (6/10), P = 0.018 and 80% (8/10) P = 0.865, respectively. The mean diameter values presented later are therefore only those participants in whom tertiary vessels were detected.The mean total number of arteries and veins was significantly reduced by 24% and 29%, respectively, in PWA compared with controls  and veins . The only discordance between the two subgroups compared with controls, was that participants with severe FH had a significant reduction (P = 0.044) of 15% less primary veins, whereas the moderate FH subgroup did not . This was mainly due to a significant reduction of 24% in the number of secondary veins compared with controls . Conversely, mean diameters of veins were significantly greater than in PWA compared with controls . This was mainly due to changes in the secondary vein subgroup .Mean diameters of primary arteries of PWA , Fig. 4 P = 0.042) and controls , the difference was more pronounced in the severe FH subgroup  whereas not significant with the moderate FH subgroup  by 13%.The PWIIN group did not demonstrate any significant differences in vessel diameter with either the control or PWA groups by vessel type or branching. However, when subdivided into quadrant, it was found the mean diameter of the arteries in the PWIIN group was significantly reduced in the superior temporal quadrant compared to controls  are presented in P values of P < 0.1 and r > 0.4.Retinal thickness measurements at a horizontal distance of 6.3 mm from the optic disc center could be derived for 18 of the 24 PWA, with motion artefact due to nystagmus being the main cause of poor data that could not be analyzed. The results of Pearson correlation analysis  are presented in The results of Pearson correlation analysis between vessel parameters and refractive error , intermediate, and deep vascular plexus.,In healthy eyes, the vascular network emerges from the optic nerve head at 16 weeks gestational age (WG) and spreads across the inner retina, reaching the temporal periphery at 40 WG.,One possible reason for these findings is reduced oxygen demand resulting from underdevelopment of the neural retinal network in albinism.In contrast to arteries, secondary venous branches were significantly thicker than in controls. We may speculate the reason for this upon their differing physiology. One key difference is that retinal arteries are lined with smooth muscle which enables autoregulation of blood flow.,,The underdevelopment of the neural network in albinism is evident from FH, which resembles the immature fovea.The IIN group displayed a similar pattern to albinism both in the number and the diameter of secondary and tertiary vessels, whose figures generally lay between that of the PWA and controls, although the differences were not significantly different in this smaller sample to either the PWA or the control group. An exception was the secondary venous quantity where it was significantly less in PWIIN than controls.FRMD7 mutations.FRMD7 mutations.FRMD7 also have abnormal optic nerve head morphology,. (2021) reported how mutations in l‑dopachrome tautomerase (DCT), a key enzyme in melanin synthesis, can produce variable phenotypes resembling either IIN or albinism.There are anatomic, clinical, and genetic links between IIN and albinism. Similar but milder structural and functional abnormal retinal development in IIN, as found in albinism, has been shown in association with As of yet, the mechanisms underlying nystagmus generation in INS remains elusive, although the presence of sensory deficits in FRMD7-related IIN along with other INS more obviously associated with afferent deficits, points to abnormal sensory inputs being a common underlying cause. It is possible that the oculomotor system responds to reduced sensory inputs during visual system development by ramping up the gain of feedback control circuits. Abnormal motion selectivity in retina in mice due to altered organization of neural networks caused by FRMD7 mutations also raises the possibility that nystagmus could be driven by abnormal inputs from the retina rather than as high gain oculomotor response to reduced inputs.A limitation of this study was that a relatively small area of the retina was analyzed. Therefore, our findings may not be representative of all retinal vasculature. Another weakness was that the study only analyzed data from 10 PWIIN and therefore may not be fully representative of this cohort as well as being insufficiently powered for statistical analysis. FH grading was carried out by only a single investigator, although inter-assessor reliability in grading FH has been shown to be very high.No correlations existed between refractive error and vessel parameters in our data nor did adjusting for refractive error change statistical outcomes. However, spherical equivalent can be taken as an approximate surrogate measure for axial length, and a more robust assessment could be achieved by calibrating fundus images based on axial length, for which data were not available for this study.On color fundus imaging, PWA and PWIIN demonstrated reduced retinal vessel quantity and arterial diameter as compared with controls, which indicates underdevelopment of the superficial retinal vasculature. Venous diameter was, however, increased. Development of the retinal vascular network is guided by the neuronal network, which is also known to be disrupted in albinism and IIN. Therefore, this opens another avenue of investigation in determining the molecular pathophysiology of INS.


Median nerve CSA varies with age, geographical location, and sex at all landmarks. A low (I2 < 25%) to considerable heterogeneity (I2 > 75%) was observed, indicating the variation among the included studies. These findings show that median nerve CSA is varying not only along its course but also in other sub-variables.Median nerve cross-sectional area (CSA) was used for screening and diagnosis of neuropathy, but few studies have suggested reference range. Hence, this systematic review was performed to evaluate a normative values of median nerve CSA at various landmarks of upper limb based on ultrasonography. PubMed and Web of science were used to search relevant articles from 2000 to 2020. Forty-one eligible articles (2504 nerves) were included to access median nerve CSA at different landmarks  inlet and CT outlet). Data was also stratified based on age, sex, ethnicity, geographical location, and method of measurement. Random effects model was used to calculate pooled weighted mean , ) at mid-arm, elbow, mid-forearm, CT inlet and outlet which found to be 8.81 mm To evaluate the morphological changes of the median nerve in peripheral neuropathy, ultrasound technology has become a popular means3. Ultrasonography allowed nerve conduction studies to be supplemented via measurements of the nerve cross-sectional area (CSA).In the long course of the median nerve through the upper limb, there are multiple potential sites for lesions or injuries to occur. These lesions could be secondary to local aetiologies like trauma and compressive masses, or polyneuropathies such as diabetes mellitus, demyelinating diseases and rheumatoid arthritis causing nerve entrapment5, whereas it is decreased in neurodegenerative conditions such as amyotrophic lateral sclerosis6. While these studies have stated the CSA cut-off for diagnosing their conditions, limited studies have suggested normative ranges of median nerve CSA which could potentially be applied for screening and diagnosis of other conditions. Therefore, this systematic review was aimed to report the normal median nerve CSA along its course at predetermined anatomical landmarks based on the reported ultrasound studies. This study also analysed the median nerve CSA in subgroup variables such as age, sex, ethnicity, geographical location, and method of CSA measurement. A common reference would be helpful for clinicians in improving screening and diagnostic methods for neuropathy of the median nerve.Ultrasonography studies have commonly compared median nerve CSA between healthy controls and patients with peripheral neuropathy, thus establishing that CSA tend to increase in carpal tunnel syndrome and diabetic neuropathy7. Using broader search terms, authors systematically searched articles from electronic databases—PubMed and Web of Science until 31 December 2020. The keywords used for the search strategy were “Median nerve”, “reference values” or “normative range”, “ultrasound imaging” and/or “ultrasonography”.This is a non-interventional systematic review of published articles from online databases to evaluate the range of median nerve CSA in the upper limb using ultrasonography. Article selection process was conducted and documented with the Preferred Reporting Items for Systematic reviews (PRISMA) and Meta-Analyses guidelines8. Given the development of ultrasound technology over the last 20 years3, empirical studies published from the year 2000 onwards were considered.Peer-reviewed studies, written and published in the English language were included. Non-English articles, case reports, conference abstracts, editorial letters, expert opinion, articles without primary datas or incomplete results were excludedArticles were screened and included for the preliminary review that encompassed the primary variable of median nerve CSA, measured using ultrasonography in healthy volunteers, including control populations, at minimally one of the five proxy landmarks: the mid-arm, elbow , mid-forearm, carpal tunnel (CT) inlet (level of the pisiform), and CT outlet (level of the hook of the hamate) Fig. . These lStudies were excluded if the mean CSA was not reported, not obtained from main trunk of median nerve with the proxy landmarks not listed or undistinguished, and/or if data was obtained from pregnant women, nursing mothers or patients with history of neuropathy or associated risk factors. The list of risk factors associated with neuropathy is reported in the Supplementary Table 9.Study subjects were assessed with minimal age of 19 years because epiphyseal closure in the longer bones of upper limb is expected to happen by this ageDuplicated records of articles were excluded, remaining records were screened by titles and abstracts according to the eligibility criteria described above. Subsequently, articles included from screening were assessed in full text for review and the reasons for exclusion were noted. The flowchart for study identification and evaluation is shown in the Fig. The studies were selected and included as per following eligibility criteria.Datas from the included articles were manually extracted and reviewed as per PRISMA guidelines. Sample size, mean, standard deviation and range of median nerve CSA for each of the anatomical landmark were recorded in Microsoft excel 2010 spreadsheet  for the analysis. Secondary variables such as age, sex, geographical location, ethnicity, and method of measurement, were also noted for further stratification of the CSA. Some studies did not report data based on ethnicity, hence meta-analysis was reported as “unspecified ethnicity” and calculations were performed based on geographical location. The methods of measuring median nerve CSA and the boundary defining the median nerve was also noted, where this information was explicitly stated in the included studies. Information on positioning of upper limb, the number of operators for ultrasonic measurements was also recorded to supplement the discussion of results.10. The data from the included articles were noted as continuous variables and the analysis was performed using mean, standard deviation and reported sample size (number of median nerves). The overall mean CSA was calculated for the articles that reported the mean value of CSA for subpopulations , using this equation—Overall mean CSA = [sum of (N × mean CSA)] ÷ sum of N, where N represents the number of upper limbs/median nerves measured11. Likewise, for the studies where the standard deviations were not provided, standard deviation was calculated manually using interquartile range, median and/or P values13.Statistical analyses were performed using Open Meta software using R console 15. A continuous random-effects model was used for the meta-analysis. The measures for all included studies were calculated at 95% confidence intervals (CI), showing the upper and lower boundary limits. Statistical heterogeneity of the pooled weighted mean was quantified using chi square 2  and I2 range, interpreted using Cochrane Handbook16. The I2 statistic measured the degree of inconsistency among the studies, indicated by the percentage of total variation effects. The thresholds for I2 were indicated as low or might not important (I2 < 25%), moderate (I2 = 30–60%), substantial (I2 = 50–90%) and a considerable heterogenicity (I2 = 75–100%)17. In addition, a Chi square test with Cochran’s Q, P < 0.10 was also considered for heterogenecity17.The frequency of variations from the included studies were computed from weighted mean estimates using DerSimonian–Lard method18. The I2 statistic, weighted mean and standard error was calculated for each subgroup(s) at 95% confidence intervals based on Cochrane guidelines17.In addition, appropriate subgroup analysis was performed to make comparisons and to identify the sources of heterogeneity. The variability was investigated based on sex, geographical location, ethnicity, and method of measurement with the datas stratified for each anatomical landmark in upper limb measuring median nerve CSA. Majority of the studies did not report median nerve CSA based on age groups, instead the overall mean age was available. Hence, for the purpose of analysis, we classified the age groups as young (19–40 years), middle age (41–65) and elderly (> 65) based on the mean age reported from the eligible studies. There were no clear scientific guidelines on the classification of age into different groups in the context of the evaluation of the CSA in human peripheral nerves such as median nerve. The age classification reported in this study is consistent in a clinicopathological study involving the gastric adenocarcinoma patients from different age groups19. This reporting is important as the poorly reported studies with lack of information may decrease the reliability, thus increase the potential risk of bias19. Studies were probed individually and judged by assessing five domains—objectives and characteristics of the study/subjects, design of the research study, methodology, description of anatomical structures and reporting of results. AQUA assessment guidelines define “low” as low risk of bias when the reported information is sufficient to reproduce. Studies that reported ambiguous and missing baseline information were considered to lack proper quality, hence they were ranked as “High” and “Unclear” risk of bias. Investigators #1 and #2 rated the included studies independently and all the disagreements were resolved through detailed discussion among the investigators.The quality of the included studies was evaluated using Anatomical Quality Assessment (AQUA) toolAll procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1964 and later versions.Informed consent is not relevant in this case as this is a non-interventional literature review of published articles from online databases.20 was excluded for the meta-analysis due to missing descriptive statistics information and hence 41 studies, reporting 2504 median nerves in 1614 healthy individuals were used for final systematic review. The number of articles that presented the mean median nerve CSA were seven31, seven29, fourteen35, twenty-seven58 and ten 57 based on mid-arm, elbow, mid-forearm, CT inlet and CT outlet, respectively. The study identification process and PRISMA flowchart outlining the systematic article review is provided in Fig. The initial literature search screened and potentially identified a total of 311 articles (158 records in PubMed and 153 in Web of Science), searched up to 31st December 2020. Subsequently, 61 duplicates were removed. The remaining 250 articles were scrutinized based on titles and abstracts. 130 papers were excluded further as their abstracts lacks the information on the median nerve and ultrasonography. From this, 120 potentially eligible articles were identified and 78 were excluded further (studies neither does not have full text nor failed to meet inclusion criteria), resulting in extraction of 42 studies meeting the inclusion criteria. Among the 42 studies, one studyThe risk was evaluated as “low” for majority of the included studies in all five domains. The summary of assessment of risk of bias is shown in Fig. 2, CI ; 8.57 mm2, CI ; 7.07 mm2, CI ; 8.74 mm2, CI  and 9.02 mm2, CI , respectively. This indicates that the median nerve CSA is variable at different points in its course, such that it decreases from the mid-arm to mid-forearm and then increases distally at the CT inlet and outlet. A significant Cochran Q statistic I2 > 90% for each landmark showed the presence of considerable heterogeneity variation among the included studies  was observed in most of the studies.A total of thirty-four studies reported the overall mean age of the participants which ranged from 21 to 82. The median nerve CSA for age groups at different landmarks were summarised in Table 52 involving 534 and 614 median nerves were reported for males and females based on different landmarks (Table 2) except for CT inlet and mid-forearm where CSA values were similar (< 0.5 mm2).Seven studies2) compared to Oceania (9.28 mm2) and Asia (9.06 mm2) studies. At elbow, the mean median nerve CSA was found higher for Europe (9.20 mm2) compared to Asia (8.27 mm2). At mid-forearm, the mean median nerve CSA appears to be larger in Americas (9.38 mm2) followed by Europe (7.15 mm2), Asia (6.24 mm2) and Oceania (5.91 mm2). Interestingly, the mean median nerve CSA for CT inlet seems to be similar for middle east (8.77 mm2), Oceania (8.71 mm2), Europe (8.90 mm2) and Asia (8.68 mm2) studies. However, the mean median nerve CSA at CT outlet is larger for Asia (9.20 mm2) compared to the middle East (8.18 mm2) reports. These results indicate that there is a considerable variation of median nerve CSA among the different geographical locations for different landmarks of the upper limb except CT inlet (< 0.25mm2).Forty-one studies reported median nerve CSA from different geographical locations. Majority of the studies (n = 29) did not report the sub-population ethnicity and hence the meta-analysis was performed based on geographical location reported in the studies Table . At mid-55 articles clearly reported the ethnicity of the sub-populations. Of this, seven articles reported for Chinese55, two for Caucasian41, two for Indians32 and one article presented for Japanese23. At mid-arm, the pooled weighted mean CSA of Chinese (9.57 mm2) is more than the Japanese (8.20 mm2). The mean CSA of median nerve appears to be smallest at elbow in Indian population (6.90 mm2), compared to the Japanese (9.10 mm2) and Chinese (8.65 mm2). Likewise at mid-forearm, the mean CSA of median nerve is higher in Caucasian (8.20 mm2) compared to the Indians (5.64 mm2) and Chinese (5.70 mm2) subjects. At CT inlet, the Caucasian (8.50 mm2), Japanese (8.50 mm2) and Chinese (8.12 mm2) CSA values were similar. The median nerve mean CSA at CT outlet (8.97 mm2) was available for Chinese subjects only. These findings represent the differences in median nerve CSA along its course of upper limb for different ethnicities , out of which one article53 included the epineurium. Two articles53 inferred to have excluded the epineurium, while studies from Bayrak et al., (2007)54 and Lu et al., (2015)55 did not provide any ultrasound images of median nerve CSA for inference55. Two studies36 used unique methods of measurement  but failed to mention if the epineurium was included or not. One article31 reported the measurement of direct tracing within the hyperechoic epineurial rim and continuous trace tool within the outer epineurial rim. In summary, direct tracing was most reported method that used to measure the CSA and this method yielded higher CSA value at CT outlet (9.24 mm2), followed by mid-arm (9.14mm2), CT inlet (8.96 mm2), elbow (8.57 mm2) and mid-forearm (6.73 mm2) compared to the other methods. Likewise, other methods such as Freehand lasso adobe photoshop produced abnormally higher CSA value in mid-forearm (11.75 mm2). In conclusion, considerable heterogeneity (I2 > 90%) was observed for this variable at all landmarks, except at CT outlet reported a moderate heterogeneity (I2 = 43.12%) when measured using elliptical selection method  among the rest of the studies included in this review. The authors included the epineurium in its CSA measurement but were unable to elucidate the contributing factors behind this variation. Lastly, the study from Tsai et al.38 did not report the results of the median nerve conduction in the control population, thus investigators were unable to determine whether the control population included individuals with subclinical median nerve neuropathy.Interestingly, the median nerve CSA was much higher at the mid- forearm59. The weighted pool estimates of median nerve CSA tends to be the smallest at the mid-forearm (7.07 mm2). This can be explained by the branching of the anterior interosseous nerve at the forearm which would further reduce the median nerve CSA in the mid-forearm before increasing again prior to reaching the carpal tunnel, as evidenced in cadaveric studies61. The distal increase of CSA could be possibly explained by the redistribution of nerve fibres into more fascicular bundles62. However, a cadaveric study by Perumal and Stringer63 showed no significant difference in the number of fascicles in the median nerve around the carpal tunnel area and the redistribution of fascicles affected the antero-posterior and transverse diameters, but not due to the CSA. Yet, the authors of the same study acknowledged the limitations such as the smaller sample size, age, and gender distribution of the samples63.Despite these anomalies, the overall measurement of median nerve CSA was reported to decrease in size, followed by an increase distally down the upper limb. The decrease of median nerve CSA in the proximal upper limb could be explained by the branching of the median nerve at the elbow as it provides an innervation to the forearm muscles, such as the pronator teres, flexor carpi radialis, flexor digitorum sublimis and palmaris longus2 which is considerably higher compared to other studies for the similar age group. This could be due to use of adobe photoshop to trace the median nerve boundary instead of tracing method in the ultrasound machine. Another reason for varied CSA for different age groups at different landmarks might be due to accumulation of inter-fascicular adipose, connective tissue, redistribution of fascicles and compartmentalisation in the median especially at CT region65. One article34 reported mean age based on body mass index, highlighting that the weight considered as major parameter for predicting median nerve CSA. Chen et al. (2011)21 compared median nerve CSA at the mid-arm, CT inlet and CT outlet among different age groups and reported no statistical difference between the middle-aged and elderly groups. Hence, considering the limited number of articles that reported age range, further research is recommended to support both the trend in CSA at these landmarks and their causative mechanisms.There is no uniform age classification to investigate the median nerve CSA changes in the normal subjects. This is a limitation to understand the progress of the median nerve CSA from young and elderly. However, we classified the age and analysed the changes in CSA based on the overall mean age reported in the studies. Except for elbow and CT inlet, the CSA was found to be varied for different age groups at different landmarks. This observation may be due to limited number of studies with sample size Table  and diffSub-group analysis based on geographical location showed a considerable variation of median nerve CSA among the different geographical locations for different landmarks of the upper limb except CT inlet. This indicates the CSA values reported in one part of the world may not be applicable to other geographical locations. So, it may be worth to have their own data of median CSA representing the country specific/ ethnicity. This sub-group analysis also suffers from the small number of available studies that explicitly reported median nerve CSA based on the geographical location and ethnicity. Therefore, future studies would be needed for determining the relationships of median nerve CSA across different ethnic groups.66, which contribute to correlate with peripheral nerve CSA67. However, with limited number of eight articles reporting median nerve CSA by sex, more research has to be conducted in future to substantiate the finding from this review. Notably, Kaymak et al.48 presented a larger mean CSA of 11.5 mm2 at the CT inlet in females, which is larger than the mean CSA noted in both the male50 and female populations52. The reason for Kaymak et al. study48 deviating from the trend discussed above is uncertain, although it could be related to the reduced reliability of this study as discussed above. If data from this study was omitted, the mean CSA at the CT inlet in females would range from 7.94 to 8.83 mm2 as reported in these three articles53.Comparison of median nerve CSA by sex indicated that males generally have larger CSA than females. A proposed reason could be males usually having greater weight and body mass index (BMI)48, and Li et al.35 who performed CSA measurements using computer Freehand lasso adobe photoshop software instead using ultrasound machine. Although there was variation in the method of measuring median nerve CSA, both direct tracing and ellipsoid formula (CSA = diameter × width × π) measurements yielded similar results68 and thus should not affect reliability of the findings in this review. On the other hand, although there was disparity in whether the epineurium was included within the median nerve CSA measurement, the significance of epineurium thickness in the comparison of median nerve CSA has hardly been discussed in current literature69. Furthermore, with ultrasonography being operator-dependant, only six articles52 reported using more than one operator and seventeen57 articles blinded their operators to reduce bias in interpreting measurements.There was lack of standardised protocol in measuring median nerve CSA across all articles. This included the various upper limb positions that were adopted for the measurement in healthy volunteers, involving wrist hyperextension reported by Kaymak et al.2 range greater than 80%, we also found low to moderate variations (I2 = 0–50%). Results from the latter could be due to the inclusion of the studies from the same research group and limited number of studies.Overall, this review analysed the mean median nerve CSA at different anatomical landmarks in the upper limbs of healthy individuals. However, it was limited by the presence of considerable heterogenicity among the included studies. This could be due to the slightly different anatomical variations among the healthy individuals and calculated across different geographical locations. Despite this observation, the source of heterogeneity was not identified even when subgroup analysis was performed based on age, sex, geographical location, and method of measurement. Although a very high heterogeneity was observed in most subgroups with an I70. The median nerve CSA was also not stratified by BMI as this review aimed to report the range of normative median nerve CSA values applicable to asymptomatic individuals regardless of BMI. Authors believed that this information may be useful in establishing a standardised reference range for the screening and diagnosis of median nerve neuropathy or determining the threshold for treatment like surgical repairs of median nerve injuries. As different studies used different scanning protocols and measurement methods  to obtain the ultrasound images, this may create bias in interpreting the measurements. Future studies may include establishing standardised protocols and methods to ensure data accuracy and repeatability when measuring median nerve CSA in various upper limb positions.Furthermore, the ranges of mean median nerve CSA calculated in this study were not categorized based on right and left hands, as other studies reported no significant bilateral differences regardless of hand dominance6, there were paucity of articles reporting the detailed ethnicities and age range for subgroup analysis, which may over- or understate the CSA range.In addition to a study with limited sample size as low as sevenLastly, a publication bias might have occurred as authors restricted the literature search to English language. This might have limited the complete inclusion of all existing ultrasonography studies.Investigations could explore whether differences in median nerve CSA with or without epineurium of the median nerve can be considered, although this difference may be statistically relevant and important. Beyond the range of mean CSA, inclusion of other methods for reviewing normal median nerve CSA cut-offs between articles could potentially explored in future research such as comparing the ratio between wrist-to-forearm median nerve CSA or wrist circumference to median nerve CSA. Moreover, given that the CSA of the median nerve that comprised of area formed by fascicular bundles and non-fascicular area, ultrasound measurements and comparison of the ratio between these areas can be studied further in order to potentially establish the causal mechanisms behind the trends noted in this review. There are no clear scientific suggestions on how to categorize age groups when measuring CSA in human peripheral nerves such as the median nerve. This work might encourage future research on determining the median nerve CSA in the age categories suggested.This study reported the pooled weighted mean of median nerve CSA along its length  as well as across geographical locations, age, sex, and method of measurements based on the ultrasonography studies. Our findings on normative CSA values may be considered as a resource for clinicians and surgeons as a cut-off point for investigating preclinical screening and diagnosing the median nerve neuropathy. However, further studies are recommended to obtain normative median nerve CSA values based on BMI across the various ethnic populations and different age groups using standardised protocols.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Rodentibacter strains. Identified as Rodentibacter pneumotropicus and Rodentibacter heylii, these isolates exhibited predominance patterns similar to those of Lactobacillus crispatus and Lactobacillus iners in the human vagina. Whole-genome sequencing of these Rodentibacter strains revealed shared genomic features, including the ability to degrade glycogen, an abundant polysaccharide in the vagina. In summary, we report body site-specific microbiotas in the pregnant mouse with potential ecological parallels to those of humans. Importantly, our findings indicate that the vaginal microbiotas of pregnant mice can be readily cultured, suggesting that mock vaginal microbiotas can be tractably generated and maintained for experimental manipulation in future mechanistic studies of host vaginal-microbiome interactions.Mice are frequently used as animal models for mechanistic studies of infection and obstetrical disease, yet characterization of the murine microbiota during pregnancy is lacking. The objective of this study was to characterize the microbiotas of distinct body sites of the pregnant mouse—vagina, oral cavity, intestine, and lung—that harbor microorganisms that could potentially invade the murine amniotic cavity, thus leading to adverse pregnancy outcomes. The microbiotas of these body sites were characterized through anoxic, hypoxic, and oxic culture as well as through 16S rRNA gene sequencing. With the exception of the vagina, the cultured microbiotas of each body site varied by atmosphere, with the greatest diversity in the cultured microbiota appearing under anoxic conditions. Only cultures of the vagina were comprehensively representative of the microbiota observed through direct DNA sequencing of body site samples, primarily due to the predominance of two IMPORTANCE Mice are widely utilized as animal models of obstetrical complications; however, the characterization of the murine microbiota during pregnancy has been neglected. Microorganisms from the vagina, oral cavity, intestine, and lung have been found in the intra-amniotic space, where their presence threatens the progression of gestation. Here, we characterized the microbiotas of pregnant mice and established the appropriateness of culture in capturing the microbiota at each site. The high relative abundance of Rodentibacter observed in the vagina is similar to that of Lactobacillus in humans, suggesting potential ecological parallels. Importantly, we report that the vaginal microbiota of the pregnant mouse can be readily cultured under hypoxic conditions, demonstrating that mock microbial communities can be utilized to test the potential ecological parallels between microbiotas in human and murine pregnancy and to evaluate the relevance of the structure of these microbiotas for adverse pregnancy outcomes, especially intra-amniotic infection and preterm birth. In particular, the laboratory mouse is widely used for in vivo experimentation evaluating microbial causes of disease  (–Lactobacillus-dominated CSTs (CSTs I to III and V) are generally perceived as being conducive to reproductive health. Conversely, the relationship between human reproductive health and the non-Lactobacillus-dominated and thus species-rich and diverse CST IV is more ambiguous  –61, and nsenii) –. These Lguous 55–, 63–65. ogresses , 51, 66,Fusobacterium nucleatum, in the amniotic cavity, which is presumably due to hematogenous transfer and can result in stillbirth or spontaneous preterm birth  Alpha diversity. Alpha diversity  of the cultured microbiota varied by atmosphere  in all body sites except the vagina  of the cultured microbiota varied in composition and structure by both body site and atmosphere ; Fig. 3,(i) Oral cavity microbiotas preferentially recovered under different atmospheres. Under anoxic conditions, cultures of oral cavity microbiota appeared to cluster based on the relative abundance of either (i) Lactobacillus (amplicon sequence variant [ASV] 3), Muribacter muris (ASV 4), and Streptococcus (ASV 32), or 2) Rodentibacter (ASV 2) and Staphylococcus (ASV 17) . Two linear discriminant analysis effect size (LEfSe) analyses were performed, one that was not restricted to a particular taxonomic classification level  and one that was restricted to the level of ASV. Hierarchical LEfSe analysis revealed preferential recovery of bacteria from the phylum Firmicutes under anoxic conditions, specifically of the genera Enterococcus, Lactobacillus, and Streptococcus , while members of the phyla Proteobacteria and Actinobacteria were preferentially recovered under oxic conditions, including the genera Rodentibacter and Rothia. Specific ASVs of each of these genera were identified in the ASV-level analysis  and included prominent ASVs 2, 3, 15, and 79 .Bacteroides and Lactobacillus ASVs as well as low relative abundances of Bifidobacterium and Parasutterella ASVs . Hierarchical LEfSe analysis revealed a large number of taxa that were cultured preferentially under anoxic conditions compared to the other atmospheric conditions . Notably, the phyla Bacteroidetes and Actinobacteria were heavily represented, as well as members of Firmicutes, especially Lachnospiraceae and Oscillospiraceae, and to a lesser extent members of the phyla “Desulfobacterota phyl. nov.” (Proteobacteria) and Verrucomicrobia. At the genus level, 13 genera were preferentially recovered in culture under anoxic conditions, including Akkermansia, Bacteroides, Bifidobacterium, Colidextribacter, Coriobacteriaceae UCG-002, Desulfovibrio, Enterorhabdus, Faecalibaculum, Parasutterella, Lachnoclostridium, Lachnospiraceae UCG-006, Muribaculum, and Rikenella. Staphylococcus was the only genus that was preferentially recovered under oxic conditions, and no genera were preferentially recovered under hypoxic conditions . The trends in the hierarchical analysis were consistent with those in the analysis restricted to the ASV level. With respect to the intestine, 18 ASVs were preferentially recovered under anoxic conditions, including Akkermansia muciniphila, multiple Bacteroides ASVs, Bifidobacterium, Lactobacillus, and Parasutterella . Bacteroides and Lactobacillus were also recovered in culture under hypoxic and oxic atmospheric conditions, but Bifidobacterium and Parasutterella were not . Rodentibacter and Staphylococcus ASVs constituted a large proportion of the cultures obtained under hypoxic and oxic conditions, yet they were not recovered under anoxic conditions . The ASV-only analysis identified only one feature as discriminant of oxic and hypoxic cultures, a Staphylococcus (ASV 17) and Bacteroides acidifaciens (ASV 26), respectively .The microbiotas cultured from the intestine under anoxic conditions were characterized by high relative abundances of several l. nov.”  (originaStreptococcus (ASV 32) and Bacteroides sartorii (ASV 28) as being preferentially recovered under anoxic conditions from the lung . No taxa or ASVs were identified as being differentially recovered based on atmospheric conditions from the vagina.The profiles of microbiotas cultured from the lung and vagina were not affected by atmosphere . However, LEfSe analysis identified Rodentibacter (ASVs 2 and 5), Lactobacillus (ASV 3), Staphylococcus (ASV 17), and Rothia nasimurium (ASV 79). Twenty of the 33 ASVs were prominent in only one body site, typically either the intestine or lung, and limited to one or two samples at high relative abundance or present in multiple samples at a low relative abundance (Table S8).Between the four body sites, there were a total of 33 prominent ASVs (defined as having an average relative abundance of ≥1% in at least one body site and atmosphere combination) . Five ASVs were prominent among all four body sites (Table S7). These five ASVs were classified as Rodentibacter was cultured from nearly all vaginal samples and at high relative abundance, yet the presence and abundance of the two Rodentibacter ASVs differed among vaginal samples. Specifically, in most mice, only one of the two Rodentibacter ASVs was abundant . In a minority of mice, both Rodentibacter ASVs were abundant. This contrasted with cultures from the other body sites, in which ASV 5 was much less common and ASV 2 was limited to recovery under hypoxic or oxic conditions, except in a few oral samples .Lactobacillus (ASV 3) was cultured from most intestinal and lung samples regardless of atmosphere, exclusively under anoxic conditions, from most of the oral samples and was highly abundant in only a single vaginal sample . Staphylococcus (ASV 17) was commonly cultured from oral and intestinal samples but only rarely from lung samples. In the vagina, Staphylococcus (ASV 17) was exclusively cultured from samples that had an abundance of Rodentibacter ASV 5; it was detected only alongside Rodentibacter ASV 2 when ASV 5 was also abundant.The prominent Rodentibacter ASVs 2 and 5 having the greatest average relative abundance in vaginal cultures  and ASV 3 (Lactobacillus) having the greatest average relative abundance in lung cultures (24.1%). Ten of these 21 ASVs were prominent in only one body site, with six being prominent only in intestinal cultures. Only one of the 21 prominent ASVs was exclusive to a single body site; ASV 106, an unclassified Actinobacteria, was unique to the lung.After bioinformatically pooling the culture data from each atmosphere by body site for each mouse, 21 ASVs were prominent in at least one body site . Three ABacteroidetes and Firmicutes were preferentially recovered in the intestine  and evenness  between the oral cavity, intestine, and vagina. Pairwise comparisons revealed that the intestine was more diverse than both the oral cavity  and vagina , while the oral cavity and vagina were not . The low alpha diversities were largely due to high relative abundances of Streptococcus danieliae (ASV 1) and Rodentibacter (ASVs 2 and 5) observed in the oral cavity and vagina, respectively . Notably, only 339 ASVs were detected in both data sets; however, both data sets had numerous ASVs not observed in the opposing data set. For each body site, more ASVs were detected in molecular surveys than culture surveys except for the lung (Table S9). Of the prominent ASVs among both data sets (ASVs with an average relative abundance of ≥1% in at least one body site from culture or molecular samples), most ASVs were detected in both data sets overall, over 90% (46/51), and at least one-half were observed in both culture and molecular data sets at each body site . Only thRodentibacter pneumotropicus by 16S rRNA gene BLAST . The genomic potential of ASV 2 was compared to that of 16 other reported Rodentibacter pneumotropicus strains for which published genomes were available. The published strains contained 1,565 core genes that were also present in ASV 2. Based on Prokka annotation of genomes . Compared to the other R. pneumotropicus genomes, the genome of ASV 2 contained 83 unique genes, of which 81 are hypothetical proteins. The two unique genes with annotated functions were identified as DNA (cytosine-5)-methyltransferase (ydiO) and serine/threonine-protein phosphatase 1 (pphA). An additional 25 annotated genes were unique to ASV 2 and its most phylogenetically similar strain P441, including a secretory immunoglobulin A-binding protein (esiB), bifunctional polymyxin resistance protein (arnA), and lipooligosaccharide biosynthesis protein lex-1 (lex1).Of the 2,384 genes present in the ASV 2 genome, 1,505 could be confidently assigned to a KEGG molecular network . The mos genomes , the panR. heylii isolate ASV 5, 1,537 of 2,474 genes were confidently assigned to a KEGG molecular network , a duplicate outer membrane protein A (ompA), and a duplicate anthranilate synthase component 2 (trpG). Compared to the other R. heylii genomes, ASV 5 contained 182 unique genes, of which 155 were hypothetical proteins. Notable genes unique to ASV 5 include those for mRNA interferase toxin RelE (relE), a duplicate lysozyme RrrD (rrrD), very short patch repair protein (vsr), enterobactin exporter EntS (entS), a duplicate endoribonuclease ToxN (toxN) found in only one other strain, and colicin V secretion protein CvaA (cvaA). A unique feature of the genome of ASV 5 compared to those of other published R. heylii strains is the presence of genes from the lsr operon, which regulates the autoinducer-2 quorum sensing pathway, suggesting that this strain may exhibit quorum sensing, which may partially contribute to the distinct community structures observed in the present study.For the  network , and as  network . CompletadhE, for metabolizing ethanol. Other features unique to ASV 2 include metabolism of nucleotide monophosphates, the amino acids alanine and proline, and the reduction of glutathione. Notably, ASV 2 is missing several enzymes involved in the tricarboxylic acid (TCA) cycle, including citrate synthase; conversely, ASV 5 is not. However, this observation was not unique to ASV 2, as these enzymes are also missing from the other published R. pneumotropicus genomes. Collectively, they encode citrate lyase, which is likely utilized as an alternative route for citrate degradation. Pathway features that are present in ASV 5 and yet missing in ASV 2 include lysine decarboxylase (needed for the biosynthesis of cadaverine), prepilin peptidase (involved in pilus formation), nitrite reductase (involved in denitrification), UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase (involved in colanic acid synthesis), and several enzymes necessary for the biosynthesis of the sialic acid CMP-N-acetylneuraminate. One interesting metabolic difference between the two ASVs is that ASV 5 contains two genes for the degradation of glycogen , whereas ASV 2 contains only one . Also, ASV 5 contains a suite of tight adherence protein genes (tadB and tadD to -G) and several, but not all, genes necessary for operation of the type IV secretion system; virB2 and virB7 were not identified in the genome. Last, although the genomes of both isolates contain the gene encoding the LuxS protein , only isolate ASV 5 carries the necessary downstream genes for quorum sensing, suggesting a substantial ecological distinction between the two isolates.Several differences in metabolic pathways were evident between the genomes of ASVs 2 and 5. As facultative anaerobes, the genomes of ASVs 2 and 5 include genes for fermentation; however, only ASV 2 has the necessary alcohol dehydrogenase gene, Shared features of the genomes of both isolates involved in interacting with the extracellular environment include genes for the Sec-SRP and Tat export pathways, lap adhesins, type VI secretion system, and the metabolism of urea. Also, although the genome of ASV 2 does not have a putative prepilin peptidase gene, both isolates contain multiple genes involved in pilus formation. While both isolates share several notable functions associated with interacting and persisting in the environment, ASV 5 has a greater capacity to interact with the environment. The more robust genome of ASV 5 and the differences in metabolism warrant further exploration, as do the number of hypothetical proteins observed in the genomes of both species. Detailed experimental studies may elucidate the mechanisms underlying the distinct colonization patterns we observed in the mouse vagina, especially in the context of ASV 5’s apparent unique quorum-sensing ability.Rodentibacter strains (ASVs 2 and 5) while using both culture and molecular approaches, indicating that the culture approaches employed herein were representative of the vaginal microbiota. Whole-genome sequencing of these Rodentibacter strains identified many shared genomic features, including the ability to metabolize glycogen, yet there were also strain-specific features, most notably a suite of quorum sensing genes exclusively observed in the ASV 5 strain.Preferential recovery of cultured microbiotas was observed between anoxic, hypoxic, and oxic atmospheres, with greater diversity of bacteria recovered under anaerobic conditions for each body site except for the vagina. The diversity of cultured microbiotas varied by body site, with the intestine having the highest and the vagina having the lowest bacterial diversity. While some variation was evident between the cultured microbiota and molecular surveys for each body site, there was a strong positive correlation between the cultured microbiotas and molecular profiles of the vagina. Bacterial profiles of the vagina were dominated by one or two distinct 2 as a terminal electron acceptor during aerobic respiration under oxic conditions  will facilitate the growth of bacteria present at this interface, which are able to grow but are typically outcompeted by other bacteria at lower (anoxic) or higher (oxic) oxygen concentrations   (Table S1). The genera within the oral microbiota often differed between studies, suggesting that environment plays a large role in the composition of the oral microbiota. This was demonstrated explicitly when the oral microbiotas of mice from different laboratories were compared . Of the  studies –83. NotaStreptococcus, Lactobacillus, Pseudomonas, and Staphylococcus . Little overlap of abundant genera has been observed among studies. In fact, one study acquired mice from two different breeding facilities, characterized the microbiotas of the lungs, and found that there were no core bacteria common to all mice and not a single bacterium was shared between the majority of mice . Howeverlococcus , 84–87.  culture . In the the lung .Lactobacillus, Staphylococcus, and Streptococcus were observed in multiple studies (Table S3). Two studies each observed mice with similar vaginal microbiotas that could be clustered into at least two community state types (CSTs). In the first study, vaginal microbiota samples could be clustered into two CSTs based largely on the relative abundance of Streptococcus (>50% in one group and ≤10% in the other) , Bacteroides, Bifidobacterium, Desulfovibrionaceae, Lactobacillus, and Lachnospiraceae in the intestinal microbiota of the pregnant mouse  was first described in 2017 , a primary carbon source in the vagina  in the human vagina during pregnancy. Whole-genome sequencing of the Rodentibacter strains dominating the pregnant-mouse vaginal microbiota here revealed the capacity to metabolize glycogen, a principal carbon source in the mammalian vagina. This capacity is also possessed by human vaginal lactobacilli. These findings suggest the existence of ecological parallels between the vaginal microbiotas of mice and humans during pregnancy. These parallels and their relevance to host reproduction warrant further investigation.The microbiota of the pregnant mouse includes bacteria shared among the oral cavity, lung, intestine, and vagina. However, variation was evident in the microbiotas across body sites. Comparisons of culture and molecular microbiota profiles indicate that culture, especially hypoxic culture, largely captured the microbiota of the vagina but not necessarily that of the other body sites. Given that the predominant members of the vaginal microbiota can be effectively cultured in the laboratory, they can be tractably used for in utero colonization hypothesis .Culture and DNA sequencing surveys of samples from the oral cavities, lungs, intestines, and vaginas of 11 pregnant mice included in our previous study evaluating the pothesis  were anapothesis . These m2, 5% CO2), and anoxic  conditions at 37°C for 7 days. Under each atmosphere, samples were plated in duplicate onto tryptic soy agar with 5% sheep’s blood and chocolate agar. Samples were also plated on MacConkey agar under oxic conditions. If bacterial growth was observed , the bacteria were collected by pipetting 1 to 2 mL of sterile phosphate-buffered saline (PBS) solution onto the agar plate and dislodging colonies with sterile and disposable spreaders and loops. These plate wash solutions  sterile swabs as a negative control for body sites sampled with a swab  and (ii) extraction tubes with no biological input as a negative control for body sites from which tissue was collected .n = 11] and blank DNA extraction kits [n = 23]) by using the Qiagen DNeasy PowerLyzer PowerSoil kit with minor modifications to the manufacturer’s protocol. Specifically, samples were added to the supplied bead tube along with 400 μL of bead solution, 200 μL of phenol-chloroform-isoamyl alcohol (pH 7 to 8), and 60 μL of solution C1. Mechanical lysis of cells was done by using a bead beater for 30 s. Following centrifugation, the supernatants were transferred to new tubes, 100 μL of solutions C2 and C3 in addition to 1 μL of RNase A enzyme was added, and tubes were incubated for 5 min at 4°C. After centrifugation, supernatants were transferred to new tubes containing 650 μL of solution C4 and 650 μL of 100% ethanol prior to addition to the filter column and 60 μL of solution C6 for elution. The lysates were loaded onto filter columns until all sample lysates were spun through the filter columns. Five hundred microliters of solution C5 was added to the filter columns and centrifuged for 1 min, the flowthrough was discarded, and the tube was centrifuged for an additional 3 min as a dry spin. Finally, 100 μL of solution C6 was placed on the filter column and incubated for 5 min before centrifuging for 30 s to elute the extracted DNA. Purified DNA was stored at −20°C until 16S rRNA gene sequencing. Amplification and sequencing of the V4 region of the 16S rRNA gene were performed at the University of Michigan’s Center for Microbial Systems as previously described . For the molecular samples, all vaginal, oral cavity, proximal intestine, and distal intestine sequence libraries met this criterion, but only five lung libraries remained . The full data set included 176 biological samples, 15 blank extraction kit controls, and 17 negative swab controls, representing a total of 1,138 ASVs.n = 15) and blank swab (n = 17) technical controls, using the adonis function in the vegan package. For each body site, the composition and structure of bacterial profiles were distinct from those of applicable negative controls (Table S11). Prominent ASVs  in either blank or blank swab technical controls are shown in Fig. S7.To confirm that the bacterial signals detected in the molecular surveys of mouse samples were legitimate, the composition and structure of the bacterial profiles of tissues and swabs were contrasted with those of blank , Streptococcus (ASV 520), and Bacillus (ASV 6), were detected as contaminants in the tissue and swab data sets. Streptococcus (ASV 11), Muribacter (ASV 4), and Rodentibacter (ASV 5), the three remaining ASVs, had average relative abundances above 1% in at least one body site from the opposing sample type , suggesting that they may be legitimate sequences, and they were not considered contaminants and were retained in subsequent analyses. To allow comparisons of the molecular data sets with the culture data sets, the decontam results were contrasted with the culture data to ensure that ASVs abundant in culture surveys were not removed as contaminants due to the fact that they were recovered via culture . ASVs classified as contaminants through decontam were retained in subsequent analyses if they were either above 1% average relative abundance in at least one cultured body site or cultured from at least five mice for a given body site. Thirteen additional ASVs met these criteria, and ultimately, 16 of the 209 ASVs identified as potential contaminants by decontam were kept in the data sets for subsequent analyses. The 193 ASVs removed from the data set are listed in Table S12. One of the five lung samples dropped below the 100-sequence-read threshold following removal of contaminant ASVs and was removed from further analysis.After establishing that the 16S rRNA gene profiles of the tissue and swab samples from the pregnant mice were distinct from those of negative controls, the tissue and swab data sets were separately analyzed with decontam  to identvegan. No differences were observed for either composition  or structure , and these samples were bioinformatically pooled by mouse identity and considered “intestinal” samples from molecular surveys. These data were contrasted with those of cultures from the midintestine.To aid comparisons of culture and molecular data sets, the molecular 16S rRNA gene profiles of the proximal and distal portions of the intestine were assessed for differences in their bacterial profiles, using the adonis function in (i) DNA extraction from bacterial isolates. The 16S rRNA gene sequences associated with ASV 2 and ASV 5 were queried against a BLAST database of 16S rRNA gene sequences from isolates recovered and preserved during the previous study , and incubated for 48 h. Colonies were then collected using sterile inoculating loops into 500 μL of sterile PBS and centrifuged at 15,000 × g for 10 min. DNA extractions were performed using a DNeasy PowerLyzer PowerSoil kit , and the first step included resuspension of the pelleted colonies in 500 μL of bead solution and adding the total resuspended volume to the bead tubes. Two additional modifications to the manufacturer’s protocol were made: (i) 100 μL of solutions C2 and C3 were combined into a single step followed by 5-min incubation at 4°C and subsequent centrifugation, and (ii) in the final elution step, DNA was eluted with 60 μL of solution C6 rather than 100 μL to increase DNA concentration. Extracted DNA was then stored at 4°C until submission (<48 h) for whole-genome sequencing (WGS).us study . IsolateLibraries were built by using the Illumina DNA Prep protocol and Nextera DNA CD indexes (Illumina). Libraries were sequenced at the Perinatology Research Branch, using iSeq 100 reagents (Illumina) with the iSeq 100 system (Illumina) and an output of 2 × 150-bp paired-end reads.Adapters from the raw sequence reads were removed by using Trimmomatic (v0.35). Genome assembly was performed using SPAdes (v 3.12.0) through the web-based Galaxy platform  with defRodentibacter type strain genomes (downloaded through NCBI GenBank’s ftp server), and secondarily with all published Rodentibacter genomes , were processed through the UBCG pipeline by using default parameters in VirtualBox. For the phylogenomic tree featuring only published type strains, Muribaculum intestinale was included as an outgroup and Muribacter muris was included as a within-family (Pasteurellaceae) outgroup. Trees were rooted on the midpoint, using FigTree (v1.4.4)  tool  for phyl(v1.4.4) .R. pneumotropicus strain P441 and R. heylii strain G1 for isolates ASV 2 and ASV 5, respectively, using Mauve multiple-genome-alignment software (v 20150226)    . The 16  1.14.5)  and proc 1.14.5)  to estabe (PGAP)  tool usie (PGAP)  for KEGGe (PGAP) . Default(i) Alpha diversity. The alpha diversities of bacterial culture profiles for each body site under each atmosphere were characterized by the Chao1 (richness), Shannon (evenness), and inverse Simpson (evenness) indices, and variation in diversity was assessed for culture profiles under each atmosphere separately for each body site. Then, after mice that had culture profiles from three atmospheres for all four body sites were bioinformatically pooled (n = 5), diversity was compared between body sites. When all three atmosphere culture profiles were compared, data sets for each body site were rarefied to their lowest read depths . When diversity between body sites was compared, each individual mouse’s pooled samples were rarefied to the lowest read depth . Alpha diversities of sequenced microbiota (molecular profiles) of the oral cavity, intestine, and vagina were also compared in the same way and rarefied to the lowest read depth (810 reads); the lung was excluded from this analysis due to low sample size (n = 4). Rarefaction was performed in R with the phyloseq package. Alpha diversities were calculated and visualized in R using the phyloseq package and labeled in Adobe Illustrator. Alpha diversities were statistically evaluated with the rstatix package in R by repeated-measures analysis of variance (ANOVA) or Friedman’s ANOVA followed by paired t tests or Wilcoxon signed-rank tests when appropriate.vegan and pairwiseAdonis2 packages in RStudio (v 1.3.1093) and R (v. 4.0.3). Nonparametric multivariate analysis of variance (NPMANOVA) was used to evaluate the composition and structure of bacterial profiles by using the Jaccard and Bray-Curtis dissimilarity indices, respectively. For comparisons where variation by mouse identity was observed, it was secondarily controlled for using the “strata” term on mouse identity in the adonis and pairwise.adonis2 functions in the vegan and pairwiseAdonis2 packages, respectively. The composition and structure of bacterial profiles were visualized with principal-coordinate analysis (PCoA) plots generated using the RAM package in R (v. 4.0.3).Statistical comparisons of beta diversities were performed using the n = 9 for oral cavity, intestine, and vagina; n = 7 for lung).LEfSe analyses were performed to identify features, taxa , or ASVs  that were preferentially recovered in different atmospheres for each body site and secondarily in each body site after bioinformatically pooling culture data from each atmosphere. To identify taxa that were differentially abundant in the hierarchical analysis, each taxonomic level from phylum to species was included for each individual ASV, when available. In assessments of bacterial profile features preferentially recovered in one atmosphere over the other two, or in one body site over the other three, only mice with cultures in all three atmospheres from a body site were included  and cladograms  were generated by using the Galaxy hub. Each taxon is indicated on cladograms when identified as a significant feature except order .vegan package in RStudio (v 1.3.1093) and R (v. 4.0.3).Mantel tests were used to determine whether there was a correlation between the structure of bacterial culture profiles and the structure of molecular profiles for each body site. Only mice with bacterial profiles in both culture and molecular data sets in a body site were evaluated. Mantel tests were performed on Bray-Curtis distance matrices, using the Heatmaps were generated using gplots and Heatplus packages in R (v. 4.0.3), and clustering of samples was performed on Bray-Curtis dissimilarity distance matrices, using an unweighted pair group method with arithmetic mean (UPGMA) in the hclust function in R.PRJNA594727). Raw sequence files and the assembled genomes of isolates ASV 2 and ASV 5 with annotations from NCBI’s Prokaryotic Genome Annotation Pipeline are available at BioProject ID PRJNA823350 under BioSamples SAMN27293572 and SAMN27294279, respectively.Original sample-specific MiSeq run files are available in the Short Read Archive from the original study by Theis et al.  (BioProj