Imaging facilities offering PET/CT and PET/MRI provide a unique collaborative environment for the medical imaging multidisciplinary team. Diagnostic radiographers and nuclear medicine technologists especially have the opportunity to not only work together but also share knowledge and technical skills, ultimately benefitting the quality of patient care.Positron emission tomography (PET) as an imaging modality has undergone considerable innovation over the past few decades. Hybrid anatomical and functional imaging has become commonplace  with improvements in imaging technology continually delivering studies with increasing diagnostic accuracy and decreasing radiation dose to the patient. More and more radiopharmaceuticals have emerged from the research sphere into clinical practice, with the traditional PET workhorse tracer  PET has experienced exponential growth over the last few decades in terms of innovative technologies and novel radiopharmaceuticals. This has fostered increasing collaboration between radiographers and nuclear medicine technologists. Indeed, for PET, the last 30 years have witnessed a meteoric rise in clinical applications. Positrons were first theorised and observed in the early 1930s, eventually leading to the development of the first human research PET scanner by the 1970s.18F‐FDG  was central. In the form of FDG was an elegant substitute for mapping the metabolic activity of the body, demonstrating how much energy the different tissues were consuming at the time the patient was scanned. Cells with higher metabolic activity, a common but not universal characteristic of high‐grade malignancy, would accumulate more FDG, and by way of a positron‐emitting 18F atom tagged into the molecule, these cells would emit the largest number of photons. With the development of the PET scanner, we could now see where in the body those cells were. For so many patients around the world, this was revolutionary.In the early clinical days of PET scanning, as it remains to this day, the radiopharmaceutical Isolated PET scanning has been all but completely superseded by the hybrid imaging technique of PET/CT, fusing the functional PET data and anatomical CT data. The superior attenuation correction and honed ability to localise the structure accumulating FDG to a specific tissue location meant that reports were issued with superior diagnostic accuracy. The developments of time‐of‐flight technology and digital scanners, as well as advances in tomographic reconstruction algorithms, have further improved spatial and contrast resolution. Most recently, total body PET/CT has started to be used more frequently in clinical practice across Australia and New Zealand providing a long‐axial scan range which allows PET data to be obtained in a single bed position acquisition. Total body PET/CT scanners allow for lower administered activity, reduced radiation dose to the patient, shortened scan times and superior image quality by way of improved signal‐to‐noise ratios.Currently, PET/MRI is in a time of flux as it emerges from the realm of research and into clinical practice in Australia and around the world. With MRI advantageous due to its superior soft tissue contrast and lack of ionising radiation, PET/MRI has found utility in paediatric patients, in the assessment of tumours in areas traditionally imaged with MRI such as the brain and hepatobiliary system, and non‐oncological applications such as assessment of inflammatory bowel disease, neurodegenerative disorders and the heart.These hybrid imaging modalities have provided an opportunity for nuclear medicine technologists and diagnostic radiographers to work together in improving the diagnosis and monitoring of various conditions. Diagnostic radiographers tend to work in fast‐paced environments in which patient contact is limited to the brief period before and after an examination. Exam times are not strict, and bookings can be quickly rescheduled make way for high‐acuity studies. In contrast, PET imaging relies heavily on regimented timing schedules accounting for radiopharmaceutical half‐life and availability. This requires the multidisciplinary imaging team to act in sync.18F‐FDG continues to stand tall as the workhorse radiopharmaceutical of PET imaging, in recent decades the number of radiopharmaceuticals available in the research and clinical space has exploded. There are targeted tracers to assess everything from amyloid plaque deposition in Alzheimer's disease, to oestrogen receptor expression in breast cancer patients and for characterisation of primary brain tumours.Whilst 68Ga‐PSMA‐11 and 18F‐DCFPyL) which served important clinical utility for staging, treatment monitoring and radiotherapy planning, with PSMA PET/CT shown to have high diagnostic utility compared with some conventional imaging methods such as bone scan and diagnostic CT.Building on discoveries in the late 1980s and 1990s, radiopharmaceuticals targeting prostate‐specific membrane antigen (PSMA) were developed through the early 2000s. PSMA is physiologically expressed in the prostate and several other organs in the body, however, its high rate of expression of primary prostate cancer and nodal disease (approximately 95%) drove its development as a specific tracer for assessment and treatment of prostate cancer.111In‐Octreotide exploiting the propensity of these tumours to express somatostatin receptors. With these tumours often being well‐differentiated and slow growing, 18F‐FDG PET/CT was of limited utility. In addition, there were inherent challenges with 111In‐octreotide in terms of spatial resolution, prolonged duration of the study and radiation dose. The clinical emergence of 68Ga‐DOTATATE in the early 2010s offered an alternative radiopharmaceutical that boasted superior lesion to background uptake, creating a more sensitive and diagnostically accurate test.Neuroendocrine tumours are represented in a small but unique subset of oncology patients. Targeted imaging in this group is not new, with the single‐photon emitting 68Ga with the beta‐emitting 177Lu, PSMA and DOTATATE pharmaceuticals soon found their way into the therapy space. Patients with previously difficult‐to‐target metastatic malignancies now had an option in which local radiotherapy could be delivered directly to the cellular level by exploiting the characteristics of the tumour cells themselves. Trials of 177Lu‐PSMA and 177Lu‐DOTATATE have demonstrated promising results compared to conventional systemic therapy for selected groups of patients with metastatic prostate and neuroendocrine cancers.In theranostics, a positron‐emitting radioactive isotope is first paired with a specific pharmaceutical to allow diagnostic imaging targeted at a molecular level. A second isotope emitting higher energy beta or alpha particles can then be paired to the same pharmaceutical to deliver targeted treatment to the affected cells. With favourable physiological biodistribution and the potential to form a theranostic pair by swapping out the positron‐emitting 68Ga‐FAPI PET/CT graced the cover of the Journal of Nuclear Medicine in 2019, exquisitely showcasing its utility in assessment of a broader range of oncology diagnoses.18F‐FDG does) opening the possibility of developing FAPI as a targeted therapy for a broad range of deadly cancers.As we write, exciting new pharmaceuticals such as those which act as fibroblast‐activation‐protein inhibitors (FAPIs) are being trialled all over the world, on the cusp of widespread clinical implementation. Images from Two articles within this issue showcase the value that PET scans have the potential to add to our patients, both in terms of innovative applications of traditional radiopharmaceuticals and the potential value of novel agents.18F‐FDG in the assessment of colorectal cancer patients has been long held, with tumour uptake traditionally assessed with visual assessment and semi‐quantitative measures such as SUVmax . Smith and colleagues present data from their pilot project, where they have considered quantitative methods for evaluating the metabolic activity within treated lesions and thereby considering the shortest progress imaging interval by which eventual response to treatment can be predicted.The importance of 68Ga‐nitroimidazole PET/CT can play in patients with difficult‐to‐treat dormant tuberculosis.68Ga‐nitroimidazole PET/CT measured the hypoxic uptake within tuberculosis lesions, considering how lesion characteristics may influence delivery of treatment agents and thereby success of the regime. This represents just one of so many novel PET tracers being applied to the clinical landscape with the potential to impact how we diagnose and monitor patients by modelling disease processes on a cellular level.The second article explores the role which The collaboration with nuclear medicine technologists and medical staff presents diagnostic radiographers with a new experience in a highly functioning, multifaceted team that is still quite young relative to the field of medicine overall. Opportunities for research in collaboration with academics, whilst being able to give meaningful guidance around radiation safety, imaging protocols and quality improvement, will offer job satisfaction and a change of scenery to the conventional radiography modalities.Combining CT or MRI with PET imaging has offered diagnostic radiographers and nuclear medicine technologists new avenues to utilise their clinical and technical expertise within the changing diagnostic and therapeutic landscape of nuclear medicine. The two professions have worked in parallel for so long – to work together in such a new and exciting field of imaging is a unique opportunity. PET/CT or PET/MR imaging is a form of precision medicine where clinical skills, efficiency and technical knowhow are only as good as one's ability to converse and empathise with patients. Radiographers are given the opportunity to learn from nuclear medicine colleagues the nuances of patient care and professionalism whilst balancing the time‐critical nature of radiopharmaceuticals. Conversely, nuclear medicine technologists can learn and appreciate the complexities of operating diagnostic computed tomography and magnetic resonance scanners.Moving forward, training standards and agreement on competency for both professions will be important to ensure this partnership between teams to deliver precision medicine does not fold under its own complexity and disagreements around ownership of roles.The authors do not have any conflicts to declare.


In contrast, cone current, assessed with cone‐driven bright‐flash ERG a‐waves, recovers within 100 ms following similar exposures, suggesting that free opsin is less able to shut off cone current. The cone‐driven dim‐flash a‐wave can be used to track recovery of cone photopigment, showing regeneration is 'rate‐limited' rather than first order. Recoveries of the dim‐flash ERG b‐wave are consistent also with rate‐limited rod photopigment regeneration . These findings agree with psychophysical and retinal densitometry studies, although there are unexplained points of divergence. Post‐bleach ERG recovery has been explored in age‐related macular degeneration and in trials of visual cycle inhibitors for retinal diseases. ERG tracking of dark adaptation may prove useful in future clinical contexts.The substantial time taken for regaining visual sensitivity (dark adaptation) following bleaching exposures has been investigated for over a century. Psychophysical studies yielded the classic biphasic curve representing recovery of cone‐driven and rod‐driven vision. The electroretinogram (ERG) permits direct assessment of recovery at the level of the retina , with the first report over 70 years ago. Over the last two decades, ERG studies of dark adaptation have generated insights into underlying physiological processes. After large bleaches, rod photoreceptor circulating current, estimated from the rod‐isolated bright‐flash ERG a‐wave, takes 30 min to recover, indicating that products of bleaching, thought to be free opsin  following bright light exposure has been the subject of study for over one and a half centuries. The upper panels represent (illustratively rather than accurately) recovery in visual sensitivity over time following a bright light exposure (‘bleach’). Various techniques have been employed to investigate this, ranging from psychophysical to electrophysiological to densitometric. Psychophysical assessments were earliest and more accessible, assessing higher levels of the visual system (conscious perception). Densitometric assessments came later and allowed direct measurement of photopigment levels. Electrophysiological measurements are somewhere in between. The years shown represent approximately the earliest year in which a deliberate quantitative investigation of recovery following bleaching was performed or published. This review mainly considers those ERG studies conducted in the last 20–25 years, and the range of insights gained from these studies (summarised in the right‐hand box). The lowest row of panels gives an example: recovery of the dim‐flash ERG b‐wave amplitude with time in the dark after a bright light exposure . The panels in this row show the response to the same strength of flash (ERG amplitude plotted against time after flash in each panel) delivered at a progressively later time in the dark following extinction of a bright background (in this case a steady‐state exposure to approximately 3000 scotopic trolands). Abbreviations: ERG, electroretinogram; ERP, early receptor potential; VEP, visual‐evoked potential. The visual system adapts by appropriately adjusting sensitivity over a remarkable range of background intensities spanning more than nine log units. Much of this adaptation occurs at the level of the retina, with many of the underlying processes occurring within the first order neurons of the visual system, the photoreceptors themselves. Whilst light adaptation is rapid, dark adaptation, the recovery of sensitivity following an exposure that has bleached a significant proportion of photopigment, is slower, and can take over 30 min for full recovery of scotopic sensitivity following a near total bleach. The kinetics of this recovery can reveal much about the cellular mechanisms in healthy individuals and potentially in patients with retinal disease.This review will first discuss briefly some of the methods used to track visual system recovery in humans following bleaching exposures, before discussing in more detail approaches that have been taken using the electroretinogram (ERG), focusing on those conducted over the last 20–25 years. The ERG represents the summed electrical response of the retina to light stimuli. Particular stimulus protocols can be devised to selectively investigate recovery of rod‐ or cone‐driven responses, and analysis of particular waveform parameters may shed light on responses of photoreceptors or bipolar cells. As the various approaches are considered, some of the insights yielded into retinal dark adaptation mechanisms will also be highlighted. The final concluding section will include consideration of potential future clinical applicability.The decline in threshold intensity at which an individual consciously perceives stimuli following bright light exposures was measured over a century and a half ago by Aubert in Figure y‐axis plots threshold elevation on a logarithmic scale, an exponential recovery would manifest as a straight line, but the recoveries do not show a uniform gradient. However, as noted by Lamb . All‐trans‐retinol is transferred to the retinal pigment epithelium (RPE) where it is converted back to 11‐cis‐retinal. The 11‐cis‐retinal returns to the rod outer segment, and combines with opsin to form rhodopsin. This is termed the retinoid cycle , and is shown in Fig. cis‐retinal is bound to cone opsin, and similar processes occur. An additional visual cycle was identified 20 years ago  and a reference electrode placed posteriorly . Broadly speaking, currents involved in neuronal hyperpolarisation and depolarisation result in negative and positive deflection, respectively. In response to a flash of light (e.g. of a strength similar to a camera flash) a negative deflection is observed lasting several milliseconds or more, followed by a positive deflection. The negative deflection is termed the a‐wave, and the positive deflection is termed the b‐wave. The a‐wave is associated largely with the hyperpolarisation of the photoreceptors ; the b‐wave relates largely to depolarisation of the ON bipolar cells . Figure The form of the response depends on numerous factors related to the stimulus , the subject  and the recording method (the types of recording electrode and their position affect the amplitude of the response). For clinical testing, the International Society for Clinical Electrophysiology of Vision (ISCEV) specifies a minimum stimulus protocol for full‐field ERG testing . However, in order to probe kinetics of retinal dark adaptation, an approach is needed whereby stimuli are delivered at multiple time intervals following extinction of a light adapting background or bleaching exposure. A bleaching exposure is one which is sufficiently strong to photo‐isomerise (‘bleach’) a significant proportion of photopigment. Such methods were employed by the older studies mentioned in the preceding section  dark‐adapted, or (2) light‐adapted to a specific background luminance (namely a white background of 30 cd mTable A very early component (latency of a fraction of a millisecond) that can be present in the ERG elicited by bright flashes deserves mention. This was first identified in intra‐retinal recordings , not necessarily in chronological order. Insights into pigment regeneration kinetics will be discussed, as well as relation to aspects of psychophysical recovery, where relevant.Thomas & Lamb  deliverecis‐retinal.Following a full bleach, they found that rod circulating current (as estimated from the a‐wave elicited by bright flashes) was undetectable in the first 5 min and took 30 min to recover to dark‐adapted levels. By interrogating responses to dim and bright flashes, they found no evidence of a change in the amplification constant after adjustment of changes in quantal catch (this term refers to the available photopigment for photoisomerisation by light) post‐bleach. They also used responses obtained on different backgrounds pre‐bleach to derive estimates of an 'equivalent background' that would give similar response amplitudes to those they recorded post‐bleach. This procedure has been termed a 'Crawford transformation' in the psychophysical literature  to minimally light‐adapt the cones. The standard white ISCEV photopic background, for comparison, is 30 photopic cd m−2.Selective investigation of cone‐driven ERG responses requires removal or suppression of rod‐driven components. Paupoo et al.  delivereThe authors delivered flashes on the rod‐saturating background before and after intense bleaching exposures. These were bright and dim flashes (measuring the a‐wave at fixed post‐flash times in both cases) to probe recovery of cone circulating current and cone photopigment, respectively. Based on the electrical responses of single photoreceptors, the amplitude of the response to a bright flash (that transiently but fully shuts off the photoreceptor circulating current) will give an estimate of the circulating current. The basis of the use of the dim‐flash a‐wave to estimate photopigment will be discussed subsequently. The bright‐flash a‐wave amplitude recovered within 30 s following an almost total bleach, and within a few seconds following a partial bleach, indicating a recovery of cone circulating current that is orders of magnitude faster than in rods following similar bleaching exposures.Kenkre et al.  investigIn the study of Paupoo et al. , the dimHowever, the flash strengths used by Paupoo et al. , though v, and the bleach level, Km, at which recovery is half the maximal rate. Mahroo & Lamb  through a resistive barrier from a larger pool, possibly within the retinal pigment epithelium , and thus terminating the action of opsin in shutting off rod photoreceptor circulating current. This rate‐limited removal of opsin could also explain the behaviour of the S2 component of psychophysical recovery. Lamb & Pugh , current returns to dark‐adapted levels within a fraction of a second following extinction of a steady‐state bleaching background. This is in stark contrast to recovery of circulating current in rods, which occurs orders of magnitude more slowly, likely owing to the action of unbound opsin in activating the phototransduction cascade and shutting off current.ERG studies of recovery of human retinal sensitivity following bleaching exposures have contributed significantly to our understanding of recovery of rod‐ and cone‐mediated vision. They have provided evidence of 'rate‐limited' recovery of both rod and cone photopigment, and have shown cone photoreceptor circulating current to be 'immune' to the presence of high levels of photoproducts such that even with 90% of the pigment bleached  pathways to cone pigment regeneration in humans is not known. Proteins involved in phototransduction (including transducin and arrestin) are translocated between outer and inner segments during light and dark adaptation . Investigation of cone ERG recovery following bleaches (reflecting cone pigment regeneration) could be informative: it is possible that this will be impaired in fundus albipunctatus due to possible impaired delivery of retinoid to cones (depending on the extent to which the non‐RPE cycle might compensate), whilst it might be expected to be intact in Oguchi disease. Patients with Sorsby fundus dystrophy  also usually have subjective dark adaptation impairment, demonstrable psychophysically. The thickening of Bruch's membrane in these patients might impair delivery of vitamin A from the bloodstream to the retinal pigment epithelium. The ERG methods described herein could provide an objective quantitative assessment of pan‐retinal rod‐mediated recovery in these patients.Monogenic diseases in which there is known to be very slow rod‐mediated dark adaptation include fundus albipunctatus and Oguchi disease. In both conditions, ISCEV standard dark‐adapted ERGs are impaired whilst light‐adapted ERGs are within normal limits; recovery of the dark‐adapted ERG can sometimes be observed following overnight dark adaptation. Fundus albipunctatus is usually associated with bi‐allelic pathogenic variants in yme Fig. . Oguchi When considering use of such testing protocols in patients and in clinical practice, the duration and potential discomfort of tests bear consideration. Delivering significant cone bleaches can involve intense exposures that might be uncomfortable, and use of flickering stimuli to track recovery can also be unpleasant. Measurement of rod‐mediated recovery, involving dimmer stimuli, is more comfortable for the subject; however, as recoveries are slower, the time burden is greater for the patient. Pupil dilatation, necessary for achieving consistent retinal illuminance, can add to discomfort, blurring vision for some hours afterwards. The size of visual electrophysiology equipment, and requirements for electrical isolation, can make testing within the space restrictions of many outpatient clinics more challenging. Portable, hand‐held devices are now available: one device incorporates a camera so that the subject's eye can be monitored, as well as a pupilometer with automated adjustment of stimulus strength so that pharmacological mydriasis is not needed  and Moorfields Eye Charity. The funding organisations had no role in the design or conduct of the research. Views expressed are those of the authors and not the funding organisations.Peer Review HistoryClick here for additional data file.


An international systematic review concluded that individuals with poor social health  are 30% more likely to develop coronary heart disease (CHD) and stroke. Notably, the two included Australian papers reported no association between social health and CHD or stroke.We undertook a systematic review and meta‐analysis to investigate the association between social isolation, lack of social support and loneliness and cardiovascular disease (CVD) incidence among people living in Australia and New Zealand.Four electronic databases were systematically searched for longitudinal studies published until June 2020. Two reviewers undertook title/abstract screen and one reviewer undertook full‐text screen and data extraction. Quality was assessed using the Newcastle – Ottawa Quality Assessment Scale.Of the 725 unique records retrieved, five papers met our inclusion criteria. These papers reported data from three Australian longitudinal datasets, with a total of 2137 CHD and 590 stroke events recorded over follow‐up periods ranging from 3 to 16 years. Reports of two CHD and two stroke outcomes were suitable for meta‐analysis. The included papers reported no association between social health and incidence of CVD in all fully adjusted models and most unadjusted models.Our systematic review is inconclusive as it identified only a few studies, which relied heavily on self‐reported CVD. Further studies using medical diagnosis of CVD, and assessing the potential influence of residential remoteness, are needed to better understand the relationship between social health and CVD incidence in Australia and New Zealand. CVDcardiovascular diseaseDSSIDuke Social Support IndexMOSS19‐point Medical Outcomes Study Social Support SurveySSISocial Support Index1“an objective state in which a person has minimal contact with others and low involvement in local community life”.is a subjective, unwelcome experience of lack of or loss of companionship”.In Australia, cardiovascular disease (CVD) dominates as the greatest cause of Australia's morbidity, mortality and healthcare expenditure.with health and social relationships interacting to influence each other, in virtuous circles or spirals of despair”.A number of conceptual frameworks illustrate the pathways between social health and health,In 2016, an international systematic review of 23 studies concluded that individuals with poor social health were 30% more likely to develop CHD and stroke.The aim of this systematic review is to investigate the association of social isolation, lack of social support and loneliness with CVD incidence among people living in Australia and New Zealand.2This systematic review and meta‐analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses2.1We included longitudinal observational data. We included studies conducted in the general population , generalisable to people living in Australia and New Zealand of all ages. We included the social health measures of social isolation, social support and loneliness. The primary outcome was incidence of CVD, obtained through self‐report or medical records.2.2We searched four databases from the earliest record to 21st June 2020 Appendix ppendix A2.3Papers were provided an ID based on the first author's last name and year of publication. Two people  undertook an initial screening of titles and abstracts independently, with discrepancies included in the full‐text screen. There were no relevant non‐English articles or conference abstracts. One author (RF) screened all full‐text papers and performed data extraction. A second author (AZZP) checked the extracted data. Two authors (RF & AZZP) independently assessed the quality of included studies using the Newcastle – Ottawa Quality Assessment Scale,2.4I2 statisticMeta‐analysis followed the Cochrane Collaboration33.1encompassed the two Australian studies included in the international systematic review.Five papers met our inclusion criteria Figure , Table 1One included paper (Simons 2013One included paper (Sahle 20203.2Comparability” category for not adjusting for our list of most important factors. Most studies lost a point in the “Outcome” category as assessment of CVD was self‐report and the follow‐up was not long enough (defined as 5‐year or more).All studies were rated as high risk of bias, with NOS scores ranging between four and seven stars Appendix . All stu3.3The five included papers reported six CVD outcomes; two studies assessed CHD  but the difference did not reach statistical significance (P > .05). There was no strong evidence of publication bias (Appendix P = .919).Across the four outcomes there was substantial heterogeneity  or data source (ALSWH or Dubbo) Figure . Further4The five included papers reported six CVD outcomes and represented three Australian longitudinal cohort studies. The included papers reported no association between social health and incidence of CVD in fully adjusted models, and most unadjusted models. Notably, all included papers were assessed as high risk of bias, mainly due to lack of adjustment for important confounders, self‐report of CVD and short follow‐up.Since our systematic search in June 2020, we have identified only one further publication that would have been included in this review. Freak‐Poli et alDespite our inconclusive findings, one excluded study provides evidence that the international patterns between poor social health and CVD are relevant to New Zealanders. Caspi et alMany people in rural and remote Australia are socially isolated, with less face‐to‐face contact with family, friends and other support networks”.in the largest urban [city] regions and in sparsely populated states and territories”.Across Australia, there was no appreciable difference in the level of social isolation [amongst older Australians] between metropolitan and non‐metropolitan regions …[However], notable variation emerges between regions when the data are mapped.”Future research needs to assess the contribution of Australia's and New Zealand's unique geographical spread, which may be masking the relationship between poor social health and incidence of CVD in the included studies. We know that people living in remote areas of Australian are more likely to have prevalent CVD and die from CVD,4.1The few papers we included provided findings that contradict the international systematic review reporting that poor social relationships are associated with a 29% and 32% increase in risk of CHD and stroke incidence respectively.loneliness and social isolation” in the title, the search terms included “loneliness, social isolation, social relationships, social support, social network”. Valtorta et alThere seems to be very little difference between Valtorta et alWe were only able to identify two slight distinctions between our systematic review and Valtorta et al4.2Our review mirrored the methodology of an international systematic review,4.3generalisable due to different social, political and cultural systems. Included papers reported no association between social health and incidence of CVD in fully adjusted models, and most unadjusted models. The included papers relied heavily on self‐reported CVD prevalence and incidence, hence further studies in Australia using medical diagnosis of CVD  are required. Additionally, further research should explore whether Australia's and New Zealand's unique geographical population spread plays a role in the relationship between social health and CVD.Our systematic review is inconclusive regarding the association between social isolation, lack of social support and loneliness and CVD incidence among people living in Australia and New Zealand. We identified five eligible papers from three longitudinal Australian cohort studies. No eligible studies were identified from New Zealand, highlighting a huge deficit in current research, as even research in Australia may not be None declared. The data collection, analysis and interpretation of data; the writing of the manuscript; and the decision to submit the manuscript for publication were solely at the discretion of the researchers, independent of funders.RF, AZZP and JH contributed to data screening and data extraction. RF and AZZP undertook quality appraisal. RF undertook calculations for meta‐analysis. RF and FB undertook critical interpretation of the data. RF wrote the manuscript. All authors contributed to, as well as, approved the final manuscript.


The Chinese Communist Party is consolidating one party rule under the leadership of Xi Jinping. Beijing seeks to rule by central mandate while limiting local autonomy. The central government response to the COVID‐19 public health emergency reinforces this view. In January 2020 Beijing established the Central Epidemic Response Leading Group to mobilise a comprehensive nationwide policy effort to contain the virus. The exceptional nature of the COVID‐19 national emergency allows the central government to project power over local authorities and leverage over citizens, but we argue that this is a short‐term phenomenon because local disease control initiatives remain important, with local authorities adapting national policies to meet constituent needs. There are degrees of policy discretion and divergence at the subnational level that enable context‐specific responses to the virus within China’s strict bureaucratic hierarchy. Primary data derives from interviews and observations in Nancun village, Hebei Province, conducted from January to April 2020. Evidence from Nancun explains how local authorities interpret the edicts and mandates of the central government. By mid‐January 2020 genomic sequencing identified the zoonotic novel coronavirus now known as COVID‐19, and asymptomatic carriers had already journeyed home from Wuhan to celebrate the Lunar New Year , in power since 1949, has further consolidated one party rule. Beijing is recentralising political authority, tightening its grip on key bureaucracies, and pushing broad national policy initiatives  and was therefore expected to strongly restrict discretion Chung, , p. 90. Our empirical findings broadly support the ‘encompassing‐governance‐urgent’ policy dimensions of an extreme public health emergency such as SARS or COVID‐19, but we argue that within a constrained, coercive environment there are local forms of policy discretion and divergence that should be documented. For a total mobilisation policy response to work, coordination with subnational units is required, and the nature of subnational level policy responses matter.This article proceeds as follows: Section 2Studies suggest that collaborative crisis management often follows the bottom‐up principle of ‘disaster subsidiarity’ with local authorities leading responses, and even in top‐down centralised systems such as China and Vietnam there is a need for central‐local coordination to manage complex emergencies , headed by Vice President Li Keqiang and deputised by Wang Huning.Hebei Province is part of the Beijing‐Tianjin‐Hebei region located in the northeast known as the ‘capital circle of China’ . Qicun township officials agreed to travel to Nancun to participate in interviews on 28 February 2020. Participants included the head of the township government , the director of the publicity department and various other officials in supporting roles. All participants in the research could speak freely, under condition of anonymity, about their experiences in responding to the COVID‐19 crisis. Interview questions focused on local response strategies and approaches, and the ways in which various groups were mobilised to contain the transmission of COVID‐19 including local authorities, Party cadres, physicians and healthcare practitioners, social organisations and volunteers. To crosscheck and give further context to these interviews, unpublished government documents and reports were examined along with media coverage of the crisis.3The responses to COVID‐19 in Hebei Province can be separated into three stages. Stage 1 is from 31 December 2019, when the outbreak of COVID‐19 in Wuhan was first reported, to 23 January 2020 when the Qicun town government was instructed by the subnational county government to monitor all migrant workers and students returning from Hubei Province . As a local leading group for COVID‐19 prevention had not yet been established at this early stage, all the track, trace and monitor work was carried out by local organisations without formal supervision from the township government. This meant that if locals failed to disclose that they had recently returned from Wuhan, the village cadres ‘would not put their fingers into another’s pie’ . Residents from Nancun recall that two university students who came back from Wuhan had been seen wandering around the village before the Lunar Festival. If the students were subsequently diagnosed with COVID‐19 ‘the whole village would be finished’ . This early‐stage response shows that in the absence of central government command, which takes precedence, subnational authorities had the flexibility to enact localised and context‐specific policy responses to the virus.Stage 2 is from 24 January 2020 to late February 2020, the most intensive period of COVID‐19 control when the town government formed a leading group to combat the virus .The comprehensive coordination group (综合协调组).The epidemic disposal group (疫情处置组).The material support group (物资保障组).The publicity guidance group (宣传引导组).The emergency prevention team (应急防控组).The supervision and inspection group (督导检查组).In China, townships are the lowest administrative units with emergency response powers (属地管理) as stipulated in the 2007 Emergency Response Law. Below the township government there is no administrative unit at the village level Wang, . The graEach group performed specific functions and cooperated with other groups to carry out core work in epidemic prevention as well as public outreach and related activities. At least 20 cadres from the Qicun township government along with local health centre staff were seconded to the seven groups, and at the time of interview ‘none of them have had a day off since’ the groups were formed . Respondents from Qicun did not disclose any budgetary information, though they did claim that they stockpiled virus prevention materials and personal protective equipment when cases of infection in Wuhan were reported.The argument that ‘formal organization charts often hide as much as they reveal about where real power lies’ in China’s authoritarian system is still valid today Saich, , p. 142.Nancun village authorities formed their own COVID‐19 response group led by the local branch Party secretary and supported by 11 village cadres, village doctors, and school headmasters at primary and secondary levels. This organisational system proved effective in part because of the 2018 implementation of ‘grid governance’ (网格化治理). Grid governance, a significant part of the CCP’s post‐2004 efforts to improve administrative efficiency, is a ‘grassroots’ multi‐actor network governance structure that operates at subnational level, where personnel assignments and public services are allocated based on grid boundaries Tang, . In 2018Following are some of the findings of our qualitative field research and the key elements of the local discretionary measures adopted in Nancun village in response to the COVID‐19 emergency during the study period.4.1In the complex and sometimes fraught process of epidemic prevention, local‐level authorities adopted a highly mobilised operational mechanism that combined the formal political resources of the state with informal resources drawn from the village constituency. The operational mechanism observed by the authors was designed to control population flows. From the outset of the COVID‐19 outbreak in January 2020, many measures were adopted to achieve this goal, such as imposing traffic restrictions, cancelling almost all Lunar New Year gatherings and celebrations, and appealing to all residents to self‐isolate by staying in their homes. The only way to achieve high levels of compliance was to ensure adequate supplies of food, medicine and basic goods to avoid panic and disorder. The local Party‐state apparatus also deployed various forms of surveillance and propaganda to manage the villagers. Village elections have, in procedural terms, improved considerably since the 1990s but governance is still often shaped by the dynamics of village politics in which elite factions vying for the office of Party secretary control resources and wield coercive power over local populations Yao, .4.2Nancun village authorities carried out a series of household inspections from February to March 2020. During each inspection, a village cadre along with a Party member or a volunteer in protective equipment surveyed the population using household registration data and reported their findings to the township government. The first public screening for COVID‐19 in February prioritised residents who had recently returned from other cities, especially Wuhan. This group of at‐risk returnees were sent to designated isolation centres and their families were quarantined at home. Village authorities decided to put warning signs on the doors of all isolating households to discourage visitors. The signs provided the contact information of the village cadres and doctors in charge of the family being quarantined . Two follow‐up investigations took place focusing on priority individuals who had returned to Nancun for the Lunar New Year celebrations. Two village‐wide screenings were also conducted in this period to crosscheck the household registry data. The aim was to confirm the number of people in each family, to record any travel that had taken place from December 2019, to identify any specific links to Wuhan (study or work related), and to screen for any flu symptoms . Nancun’s grid governance model is contentious from a privacy and freedom perspective Zhao, , though 4.3By 25 January 2020, all 21 villages in Qicun town had imposed travel restrictions and blocked traffic, leaving only one entry and exit checkpoint to restrict access in each village. Nancun opened two corridors with checkpoints connecting with other villages. In the Chinese tradition, 25 January is an important time to visit relatives after the Lunar Festival. When the traffic blockade was imposed on such an auspicious date, village residents quickly grasped the seriousness of the epidemic . There was naturally some discontent; however, public acceptance of the lockdown may have been aided by memories of the 2003 SARS outbreak .Traffic checkpoints were highly visible, with detailed signposting and uniformed staff equipped with infrared thermometers, disinfectant sprayers, tables and chairs, tents, pillows, bedding, provisions, electric heaters, and other materials. At each checkpoint there was a village cadre and a Party member or volunteer on duty 24 hours a day. Township level cadres inspected the village checkpoints periodically every day. If they found that no one was on duty at the checkpoint, the village committee—which was responsible for upholding lockdown rules and accountable for any outbreaks—faced disciplinary measures. During this stage of the COVID‐19 response residents from other villages were not permitted to enter Nancun. Residents of Nancun who wished to travel had to follow a strict system of ‘checking, asking and registering’ (一查二问三登记) before receiving a travel permit from the village committee . The only grounds for obtaining a permit was to visit a hospital, and applicants needed a letter from their physician confirming their health condition.4.4Village authorities immediately reported any people returning to the village to their superiors at town level. From January to April 2020 villages were also required to complete daily reports by 11am and return the information to the Qicun town government, with emphasis on arrivals from Wuhan. Failure to disclose and report was considered a serious dereliction of duty. If the village was found to have deliberately concealed information, the Party branch secretary and their deputies faced automatic dismissal. If the relevant data was not reported in a timely manner, village cadres received a warning and may have faced disciplinary measures . The authors learned of at least one case of immediate dismissal for failing to report. The head of Qicun township received news of an arrival from Wuhan early one morning, but when he contacted the Party secretary in the village concerned to check the details, he found that the secretary knew nothing about the matter. This suggests that the township government had set up an additional information gathering network for real‐time monitoring of the changing situation. The head of the township government confirmed that ‘we have our own information channels’ , which indicates that the township created a new network to gain detailed information rather than relying on the reports from village authorities and cadres. The village‐level Party branch secretary was immediately dismissed for failing to report a person returning home from Wuhan .Expulsion from the Party also means losing one’s cadre status. In rural China, the Party branch secretary oversees the rural collective economy, deciding how to distribute land and manage rural enterprises, which can be very lucrative Zhang, . In addi4.5At the start of the outbreak the government rented hotels and student dormitories and converted them into quarantine centres. People returning from Hubei Province were directed to isolate at a designated quarantine centre for 14 days and then self‐isolate at home for a further 14 days. Village doctors were responsible for preliminary identification, and after reporting symptomatic patients and suspected COVID‐19 cases to the town government, the health centre arranged special medical staff and vehicles to transfer patients to the quarantine centre for 14 days. Only then did testing and tracing begin. Once discharged, people isolated at home with their family for an additional period of 14 days. The town health centre provided thermometers, disinfectants and supplies for the village doctors, and village cadres distributed a list of precautions and guidelines for families in isolation. For instance, isolating households received an information chart (信息表) to record their physical condition every day, as well as a service card (服务卡) to request provisions and essential items needed during lockdown. Village cadres worked with the local police to ensure the safe distribution of goods. The wellbeing of families in isolation was also given consideration, with counselling services ostensibly offered by village authorities, but there is no data available about this service.4.6One formidable challenge during lockdown restrictions in the first two months of 2020 was ensuring an adequate supply of medical resources and staple goods. Supply chain disruptions bring the risk of scarcity and inflation, leading to fears of public riots. Some villages in Qicun experienced price hikes in mid‐February 2020, and in one example when the price of flour suddenly increased in a supermarket locals saw this as a sign of shortages and started panic buying. The town government quickly intervened and revoked the store’s business licence, sending a clear message to other local supermarkets which then maintained pre‐lockdown prices for staple products .On 17 February 2020 the town government announced a plan to ensure the supply of daily necessities within their jurisdiction. Qicun authorities had access to the county distribution centre that was temporarily set up to meet demands for medical supplies, sanitary products, and food. The township appointed a logistics team to work directly with 10 supermarkets in their jurisdiction while liaising with county distributors and village supermarkets. Travel restrictions remained in place, but the town government created a ‘green channel’ (绿色通道) for the supply of daily necessities. Staff from one supermarket recalled that daily shipments reached the villages, with designated delivery drivers approved by village cadres supplying stores to limit physical contact .A degree of market intervention was required during the epidemic. Apart from pharmacies and supermarkets that supply essential goods, all other businesses faced temporary closures. Pharmacies and supermarkets that operated during the lockdown were granted special permission from the town government. Shop entrances had screening and testing tables to check the temperatures of customers, and a record was kept of all customers so they could be identified and contacted in the case of an outbreak. All shoppers were required to wear masks. The town government implemented a weekly market monitoring system to try to maintain the supply of essentials and manage the demand for grain, meat and vegetables. Supermarkets were advised to replenish goods to avoid panic and the impression of shortages . Pharmacies in Senzhong and Qicun were tasked with the daily monitoring of the market supply and demand for preventive equipment such as masks, thermometers and disinfectants. The town government supervised the prices and inventory of necessities to ensure stability. Staff from one village supermarket reported that in February people posing as regular customers casually enquired about the prices of various commodities, only to report suspected price hikes to the town government. At the time of research there were stories and rumours of supermarket closures in Qicun township .5The leading groups working to combat COVID‐19 continue to follow the basic outlines of the government’s organisational structure. Since May 2020, there has been a redeployment of staff and some reorganising of processes and procedures, but the epidemic response groups and their constituent members are today still upwardly accountable to the relevant government departments. After the COVID‐19 outbreak there was a risk of panic spreading among communities; ‘pestilence is coming’ became a common refrain used by village elders in their daily conversations, referencing the horrors of the plague . In the era of smartphones and instant communication, villagers can quickly disseminate news of varying degrees of reliability about the spread of infections. Doctors in the township health centre were equipped with protective clothing, goggles and masks when transferring symptomatic patients, and while these were preventative measures the optics of these scenes caused panic among some villagers who assumed that patients were infected with COVID‐19 .On 18 February 2020 stories began to circulate in Nancun village about a student who returned from Wuhan and still had a fever after 14 days of isolation. As the news of this unconfirmed COVID‐19 case spread, the severity of the epidemic grew in people’s minds. People in Qicun town began to phone relatives in Nancun to find out whether the rumours were true . Tensions rose as Nancun village went into total lockdown; the streets were empty, and families began to accuse and blame each other of breaching the lockdown rules, for example going outside to play mahjong . It later emerged that one resident who visited a doctor in Cangzhou City, Hebei Province, before the Lunar New Year and self‐isolated after returning to Nancun on 18 February 2020 had been visited at home by some villagers who were unaware of the situation at first and had inadvertently broken the rules .The false alarm in Nancun meant that neighbouring villages such as Beicun took even greater COVID prevention precautions and residents avoided anyone from Nancun, even close friends and relatives . The Party secretary of Nancun took the pragmatic view that ‘there is no need to dispel the rumours surrounding the village’ because, in the short term at least, it makes it ‘easier to prevent people from coming to our village’ . Within the village the opposite approach was taken by cadres as the rumours were damaging social relations and undermining trust. Village authorities requested permission from the town government to publish the personal information of families in isolation. The response from Qicun was that there were legal concerns, although as the purpose of publishing the private information was to protect the public during an emergency, it was therefore deemed permissible . Following this decision all information related to people in isolation was published in an official village WeChat group.Township authorities spent considerable time and energy trying to contain the threat of disinformation during the peak of the COVID‐19 outbreak. Qicun officials routinely checked epidemic information broadcasts, inspected public spaces to ensure public health bannersDuring the SARS outbreak in 2003, many villagers in Nancun questioned the lockdown policy. But during the COVID‐19 emergency response the village committee did not face much opposition—many villagers in Nancun supported the lockdown approach. From January to April 2020, the researchers only learned of one breach of the COVID‐19 lockdown. An intoxicated man from Nancun found his way to neighbouring Beicun village and was apprehended by police. The next day Nancun residents spoke with disapproval about the matter and showed little sympathy for the man who was in police custody. Access to medicine was a different matter, however, with villagers showing a greater degree of flexibility, and some rule bending occurred. Relatives of village doctors, who are responsible for managing pharmacies and controlling prescriptions, seemed to be able to get easy access to medicines and a grey market emerged .6China’s effective containment strategy for COVID‐19 explains the sub‐exponential growth of cases .Village doctors rallied to the cause, being motivated to participate in the epidemic prevention effort without any additional financial support. In China’s villages there are many so‐called barefoot doctors (赤脚医生), a reference to part‐time paramedics trained in simple techniques of diagnosis and treatment who work as unpaid volunteers under no obligation from the government and village committee. From observations in the field, it was clear that village doctors participated on the front line of the fight against COVID‐19, including monitoring and recording temperatures of people in isolation even when there was no personal protective equipment available . The Party branch secretary claims that ‘these doctors were reluctant to attend village meetings in the past; however, this time was different, they joined all the video meetings without hesitation’ . The village doctors’ physical and online participation stemmed from a moral responsibility to the community.The novel response to COVID‐19 is further evidence by villages exercising a certain degree of autonomy over policy interpretation. During lockdown, the township government stipulated that village residents can only travel ‘for major events’ . At first the village leading groups did not know how to determine what a major event was, and simply prohibited everyone from leaving Nancun. Upon reflection it was decided that only sick villagers with certificates from their doctor could leave the village to seek further testing and consultation at hospital, and should provide a hospital certificate on their return. The practical decision to allow travel exceptions on medical grounds was subsequently adopted by neighbouring villages, followed by the Qicun township government .Rural lockdown measures were strict and widely adhered to, though not all the directives of the township government were enforced. Cases of corruption and collusion occurred during lockdown. For instance, one mah‐jong hall in Nancun, whose owner was a close friend of the deputy director of the village committee, remained open in February 2020. When the township government came to routinely inspect the village, the deputy director, who was known to play mah‐jong every day except when he was on duty at the village checkpoint, warned the mah‐jong hall so that it could close before inspectors arrived . By contrast, the Party branch secretary Gu Xingkai worked tirelessly during the peak of the COVID‐19 crisis. Secretary Gu’s only son died unexpectedly before the Lunar New Year (unrelated to COVID‐19), and news quickly spread of his personal tragedy as well as his unwavering commitment to his duty. He became a symbolic figure in the fight against the epidemic and received significant media coverage throughout Hebei Province ; intensive surveillance period, with a formal leading group established by the township and village government to combat the virus (24 January to end February 2020); and easing of lockdown restrictions, when the 21 villages of Qicun township recorded zero infections (March to April 2020). Local authorities had managed the influx of returnees from various parts of China during the Lunar New Year celebrations.This rapid containment is a significant indicator of the importance of local responses in the fight against COVID‐19, where constrained but still significant local discretion exists in most major policy realms. Local resistance to central mandates in China, resulting from ‘mismatched decentralization’ policies (van der Kamp et al., No conflicts to declare.


Fetal bone retention is a rare but under‐diagnosed complication after abortion. If left untreated, it can cause menstrual dysfunction and secondary infertility. We present a case of a 39‐year‐old woman who undergone abortion 20 years ago but suffered with secondary infertility due to retained fetal bone. Retained fetal bone is a rare complication for abortion and can cause secondary infertility. There is currently no comprehensive guidance in abortion care in the UK regarding this complication, hence we call for awareness and development of guideline. Her pain was non‐cyclical, spasmodic, and often had a sudden onset. She had recurrent herpes infections for which she took acyclovir when needed, but she was otherwise fit and well. She had no history of drug allergy. She was an active smoker, smoking 1–2 cigarettes/day, and she drank 20 units of alcohol per week. Her body mass index was 31.8. A previous obstetric history revealed a mid‐trimester abortion 20 years ago because she was not mentally and physically ready to carry on with the pregnancy. The abortion was performed with surgical dilatation and curettage at 20 weeks. Transvaginal ultrasound investigation showed heterogeneous echotexture in the anterior fundal wall containing a cystic area 8 mm in size  in situ. Some research has suggested that the fetal bone may have a direct toxic effect on the developing embryo, thereby causing infertility.Currently, hysteroscopy is used in most cases as the treatment of choice to retrieve a foreign body to treat infertilityDr J Cao has participated in the care of the patient, researched and written the case report. Dr A Gumma has participated in the care of the patient, initiated and reviewed the case report. Dr C Grub has participated in the care of the patient and reviewed the case report. Mr M Khurshid has participated in the care of the patient.None.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


With the advancement of single‐cell separation techniques and high‐throughput sequencing platforms, single‐cell RNA‐sequencing (scRNA‐seq) has emerged as a vital technology for understanding tissue and organ systems at cellular resolution. Through transcriptional analysis, it is possible to characterize unique or rare cell types, interpret their interactions, and reveal novel functional states or shifts in developmental stages. As such, this technology is uniquely suited for studying the cells within the human ovary. The ovary is a cellularly heterogeneous organ that houses follicles, the reproductive and endocrine unit that consists of an oocyte surrounded by hormone‐producing support cells, as well as many other cell populations constituting stroma, vasculature, lymphatic, and immune components. Here we review studies that have utilized scRNA‐seq technology to analyze cells from healthy human ovaries and discuss the single‐cell isolation techniques used. We identified two overarching applications for scRNA‐seq in the human ovary. The first applies this technology to investigate transcriptional differences in oocytes/eggs from patients undergoing in vitro fertilization treatments to ultimately improve clinical outcomes. The second utilizes scRNA‐seq for the pursuit of creating a comprehensive single‐cell atlas of the human ovary. The knowledge gained from these studies underscores the importance of scRNA‐seq technologies in unlocking a new biological understanding of the human ovary. Next‐generation RNA‐sequencing has been widely used to profile the transcriptome of bulk tissue. Now, due to the advancement of single‐cell collection methodologies, it is possible to determine gene expression at the single‐cell level by measuring an individual cell's transcriptome. This process, known as single‐cell RNA‐sequencing (scRNA‐seq), combines high‐throughput sequencing platforms with bioinformatics analysis to provide a readout of cellular transcriptomes. The single‐cell transcriptome is a molecular signature that encompasses the genes transcribed by each cell and their level of transcriptional output. Cellular identity is more broadly defined by a variety of factors influencing the cell at any given time such as its environmental milieu, cell cycle stage, position in a developmental lineage, and external stimuli . In this method, ovarian tissue is enzymatically digested and individual cells of interest are manually collected by micropipette and placed directly into lysis buffer in preparation for sequencing. The DCL method is used in the human ovary because it overcomes a few major challenges of scRNA‐seq involving the isolation of individual oocytes due to their size and rarity. One challenge has to do with the channel diameter of droplet‐based microfluidic devices typically used to separate a bulk suspension into individual cells before sequencing. The leading manufacturing company of single‐cell sequencing devices, 10× genomics, reports that cells larger than 40 µm can clog their devices , that facilitates meiotic resumption and retrieval of matured MII eggs in clinic  in DNA. With its higher expression in the rIVM eggs, this suggested a possible compensatory mechanism for an increase in DSBs in the in vitro environment. They conclude that energy metabolism and proper mitochondrial function are the limiting factors in proper rIVM egg developmental potential and targeting these processes could be an option to improve outcomes of rIVM egg use in clinic.In 2019, Zhao et al.  and GATA‐binding factor I (GATA‐I) where expression of signaling genes in these pathways was reduced in rIVM eggs. The genes controlled by these pathways are involved in energy metabolism and antiapoptosis. Therefore, reduced regulation of these two signaling pathways in rIVM eggs might affect egg metabolism and stress responses. The authors of this paper compared their data set with that of Zhao et al. and Ye et al. (discussed above). Contrary to Zhao's group, they found that expression of ACAT1 and HADHA was elevated in the rIVM eggs compared to IVO eggs. Their data overlaps with Ye et al.'s work which showed that mIVM eggs demonstrated an upregulation of mitochondrial translation activities which was also seen in the rIVM eggs of this study. Overall, A. W. T Lee's research group revealed specific genes that could reduce the performance of rIVM eggs. They conclude by arguing the need for IVM media that is optimized specifically for rescue IVM in oocytes without surrounding cumulus cells before incorporating this technique in an IVF practice.Most recently, A. W. T. Lee et al.  performeIn summary, although the results from these three reports differed with respect to which genes were upregulated or downregulated, overall, there was a consensus that energy metabolism was dysregulated in eggs that underwent in vitro maturation.2.4Advancing maternal age, which is clinically defined as 35 years old or older, contributes to a decline in oocyte and egg quantity and quality  term among the genes downregulated with maternal age was catalytic activity whereas in genes that were upregulated with age there was an enrichment in transcriptional activation.The first of these studies utilizing scRNA‐seq to look for maternal age effects was performed by J.‐J. Zhang et al.  whose grFollowing this study, Yuan et al.  interrogND1 was downregulated with age. This gene is involved in electron transport chain in mitochondria and agrees with the overall decrease in oxidative phosphorylation and ATP synthesis seen in the previous data set by Yuan et al. Another finding consistent with the study from Yuan et al. is aging had an impact on the transcript representation of genes related to chromosome segregation. Finally, researchers found genes related to oxidative stress protection to decrease with age in both populations.The final study investigating aging was by Llonch et al. . Their sThese three data sets provide important transcriptomic factors that are altered in oocytes and eggs with advancing age. All three indicate that changes in transcriptional activity may be dysregulated in older eggs. Additionally, eggs from older patients showed impairments in oxidative stress, DNA damage repair, and chromosome segregation which suggests an impaired ability to combat DNA damage.2.5Fertilization fails in 5%–10% of IVF cycles and in 2%–3% of intracytoplasmic sperm injection cycles (Mahutte & Arici, To understand the gene regulatory dynamics involved in oocyte maturation Yu et al.  generateTo clarify the molecular mechanisms of fertilization failure at the single‐cell level, L. Suo et al.  profiledIn summary, these findings together suggest the importance of increased transcription of meiosis and cell cycle‐related genes during oocyte maturation to prepare for normal fertilization.2.6In addition to its clinical application, single‐cell sequencing technology is used to create a complete cellular map of ovarian cell types (Ernst et al., Li et al.  performeFOXO1, the downstream transcription factor of the PI3K/AKT pathway, was significantly upregulated in primordial oocytes, whereas EIF4E, a critical oocyte development gene was upregulated in primary oocytes (Ernst et al., To describe the gene activity and canonical pathways of oocytes in the primordial to primary stage transition, Ernst et al.  utilizedC1S in healthy compared to degenerating follicles and an overabundance of CD68+ macrophages in degenerating follicles suggesting an innate immune response potentially involved in follicular remodeling. Other complement system genes they characterized included C1R, C7, and SERPING1 which reflect immune cells resident to the adult ovary.Fan et al.  utilizedTo understand the ovarian single‐cell landscape more completely, Wagner et al.  combinedCD99, CD55, ENG, and ANPEP, that distinguish granulosa cells from theca cells (Man et al., PVRL1 distinguishes cumulus versus mural granulosa cells—the two granulosa cell compartments within the antral follicle (Man et al., Several other sequencing papers have tried to elucidate the transcriptomic signatures of oocytes and their surrounding somatic cells across a range of follicular developmental stages (Fan et al., These papers have contributed greatly to our understanding of the cell types present in the human ovary and the molecular signatures of unique compartments and follicle stages. However, scRNA‐seq in the human ovary has some limitations that are demonstrated in these papers. While extremely beneficial, human tissue studies are limited by donor tissue availability and small samples sizes. Some of the patients whose tissue was analyzed were cancer patients undergoing elective ovariectomy (Fan et al., 3The emergence of robust sequencing platforms and advanced single‐cell isolation techniques has provided the foundation for exploring the transcriptome of single cells in the human ovary. So far, applications of scRNA‐seq have focused on improving IVF outcomes and creating a comprehensive map of ovarian cell types. Now, there are a plethora of enriched gene datasets and pathway analyses to investigate as potential therapeutic or experimental targets. Due to the limited availability and precious nature of human tissue, there remains a need for repeated experiments with larger experimental groups. These reviewed single‐cell sequencing studies are limited further by their inability to deduce transcriptomic shifts through spatial relationships in the tissue. Additionally, due to the heterogeneity of the human ovary, there is an increased risk of missing unique or under‐studied cell types in sequencing experiments which is influenced by the amount of tissue received and the area within the ovary the tissue was taken from. A promising solution to these limitations involves using spatial transcriptomics methods, such as Visium or nanoString, which provide the transcriptome data of groups of cells in specific locations in the native tissue Marx, . This meJordan H. Machlin: Writing–review and editing; writing–original draft; Investigation. Ariella Shikanov: Writing–review and editing.The authors declare no conflict of interest.


The central nervous system (CNS) coordinates all our body functions. Neurons in the CNS parenchyma achieve this computational task by high speed communication via electrical and chemical signals and thus rely on a strictly regulated homeostatic environment, which does not tolerate uncontrolled entry of blood components including immune cells. The CNS thus has a unique relationship with the immune system known as CNS immune privilege. Previously ascribed to the presence of blood–brain barriers and the lack of lymphatic vessels in the CNS parenchyma prohibiting, respectively, efferent and afferent connections with the peripheral immune system, it is now appreciated that CNS immune surveillance is ensured by cellular and acellular brain barriers that limit immune cell and mediator accessibility to specific compartments at the borders of the CNS. CNS immune privilege is established by a brain barriers anatomy resembling the architecture of a medieval castle surrounded by two walls bordering a castle moat. Built for protection and defense this two‐walled rampart at the outer perimeter of the CNS parenchyma allows for accommodation of different immune cell subsets and efficient monitoring of potential danger signals derived from inside or outside of the CNS parenchyma. It enables effective mounting of immune responses within the subarachnoid or perivascular spaces, while leaving the CNS parenchyma relatively undisturbed. In this study, we propose that CNS immune privilege rests on the proper function of the brain barriers, which allow for CNS immune surveillance but prohibit activation of immune responses from the CNS parenchyma unless it is directly injured.  The immune system has evolved to protect the body from attacks from microbial pathogens and trauma and is thus ultimately tuned to ensure host survival in a hostile environment , 2. The 3, respectively, to imprint gut homing and skin homing trafficking programs as well as site‐specific effector functions in naïve lymphocytes (summarized in [During their activation naïve lymphocytes are imprinted with trafficking programs (expression of a combination of adhesion and chemoattractant receptors) that—once they have via the efferent lymphatics reached the bloodstream—ensure their migration back to the tissue drained by the respective lymph node and thus direct site‐specific adaptive immune responses. For example, in gut and skin draining lymph nodes DCs have been shown to play an important role in this process by processing food‐derived vitamin A and ultraviolet‐induced vitamin Drized in ). With tRM) cells expressing the T‐cell activation marker CD69 and the αE‐integrin subunit CD103, which when associated with the β7‐integrin subunit forms αEβ7‐integrin, a ligand for E‐cadherin, the adhesion molecule forming epithelial adherens junctions [RM cells. Systemic cellular immune memory is realized by central memory T (TCM) cells that re‐express L‐selectin and the chemokine receptor CCR7—the trafficking program of naïve lymphocytes—allowing TCM cells to recirculate through lymph nodes throughout the entire body, where they can then provide accelerated immune responses to their cognate antigen even if encountered at a different anatomical entry site  and spinal nerves . However, the CNS resides behind blood–brain barriers (BBBs) that restrict pathogen and immune cell entry from the periphery into the CNS parenchyma and also lacks lymphatic vessels. The CNS must thus have a unique relationship with the immune system that differs from that of peripheral organs.Indeed, experiments performed originally by Shirai 100 years ago  and follIronically, this concept of “CNS immune ignorance” literally “ignored” the additional observations of Medawar , which sThe brain barriers simultaneously maintain homeostasis of the CNS parenchyma while enabling immune surveillance at the CNS borders Fig. . The outglia limitans and establishes a second barrier “behind” the BBB and BCSFB, which in the healthy CNS prohibits immune cell entry into the CNS parenchyma . Thrized in ). While rized in , CNS borThere are no tissue‐resident T cells or B cells in the CNS parenchyma of healthy young adults, but there are low numbers of immune cells found in the CSF and these are dominated by T cells with small numbers of monocyte‐derived dendritic cells (mDCs) and CSF‐specific monocytes combining transcriptional signatures of monocytes, CNS border‐associated macrophages, and microglial cells , 35, 36.As if this immune protection was not enough, the CNS has recently been referred to as immune‐fortified at its outer borders , where tIn addition to blood‐derived immune cells, the dura mater may have access to immune cells directly originating from the bone marrow in the adjacent skull or vertebrae of the spine , 40. TheThe vast majority of studies addressing the anatomical routes as well as cellular and molecular mechanisms of immune cell migration into the CNS have been performed in the context of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS) . By the Unless they recognize their cognate antigen on perivascular or leptomeningeal APCs—the “castle moat sentries”—these activated T cells will, however, not cross the glia limitans , 53 and A study following the trafficking of fluorescently or radioactively labeled lymphocytes collected from the efferent lymph in sheep has questioned the requirement of recent activation for lymphocytes to reach the CSF . This reThe migration of T cells across the endothelial brain barriers is a multistep process regulated by the sequential interaction of different signaling and adhesion molecules on the CNS endothelium and the T cells and takes place at the level of subarachnoid and subpial venules or parenchymal post‐capillary venules . AccountStudies in the context of EAE have furthermore suggested that while Th1 cells preferentially infiltrate the spinal cord using α4β1‐integrins , 69, Th1Recent studies have also suggested that immune cells localized in the dura mater may reach the CNS from this site , 40, 45.Once they have reached the subarachnoid space, elegant in vivo imaging studies have shown that activated T cells crawl on leptomeningeal cells in a random Brownian walk using α4β1‐integrin and LFA‐1 and eventually encounter border‐associated macrophages or would be flushed away by the flow of the CSF . RecogniUnder physiological conditions, the glia limitans provides a barrier for the migrating immune cells, which in the absence of neuroinflammation do not enter the parenchyma . AstrocyWhile a vast majority of studies have focused on investigating the migration of defined T‐cell subsets like Th1, Th17 or, to a lesser degree, CD8 T cells, there is not much knowledge on how the site of priming affects T‐cell migration into the CNS. Studies where EAE is induced by the adoptive transfer of T‐cell blasts have proposed the lung as a tissue licensing T cells to preferentially migrate to the CNS . Howeverbona fide lymphatic vessels within the brain and spinal cord parenchyma and the dual‐walled barrier anatomy of the CNS, it is clear that unique anatomical arrangements must exist for fluid and its components and especially for immune cells to reach the peripheral lymphatic system.Research from the past few decades has shown that the traditional concept that lymphatic vessels are either not involved in or had only a minor contribution for CNS drainage no longer holds true . Classicfila olfactoria) are covered with sheaths of connective tissue that forms a fluid space that is considered to be continuous with the subarachnoid space. It is thought that CSF is driven into this space due to hydrostatic pressure gradients where it then gains access to the nasal mucosa interstitial tissue or perhaps even directly to lymphatic vessels within this tissue [Several cranial nerves emerge through foramina at the base of the skull and routes along these nerves have been repeatedly implicated in CSF outflow since an initial report in 1869 by Schwalbe , 95. Of s tissue , 96. Thes tissue , 98, 99.It still remains to be determined how intact the arachnoid or perineural layers of tissue are at this location and thus whether any barriers might exist for fluid, solutes, or cells to reach the lymphatic system. It has been proposed that barrier properties are no longer present once the nerves have penetrated through the cribriform plate, as the perineural layers become loosely adhered to the nerves with discontinuous junctions between cells , 100. InSeveral studies have also demonstrated outflow of CSF tracers along with the perineural space that accompanies the optic nerves as they exit through their respective optic canal , 107–109filum terminale [Compared to the cranial pathways, a lesser proportion of CSF is drained from the spinal column. Much focus has been on the regions at the dorsal nerve root, where the spinal nerves emerge from the CNS to the periphery and are accompanied by layers of the meninges in a similar manner to cranial nerves. These layers form what is known as the subarachnoid angle or cul‐de‐sac at the dorsal root ganglion . Lymphaterminale , consisterminale , 118. Aferminale .A network of lymphatic vessels in the dura mater was rediscovered in two studies published in 2015, triggering much research activity on this topic , 94. TheAlthough these dural lymphatic vessels have been the subject of intensive research since their rediscovery, several questions remain regarding their role in efflux from the CNS. For one, their location has been firmly established to be within the dura mater layer of the meninges. This is significant, as the dural blood vessels lack the endothelial cell barrier properties that are found in the CNS parenchyma and, thus, this places the dural lymphatic vessels outside the arachnoid barrier isolating the CNS from the periphery . Second,Even more unsettled are the potential routes for the interstitial fluid (ISF) of the CNS parenchyma to clear to the periphery Fig. . The traInvestigators have also proposed that CSF can enter the brain through anatomically not precisely defined paravascular/perivascular routes through a bulk flow mechanism. Traditional evidence for this has been based on postmortem assessments of tissue, in which tracer can be found at a perivascular location surrounding parenchymal blood vessels, even with short periods of time between the time of injection and death of the animal , 137. HoAt this point, it is important to note that experimental and modeling studies have raised many issues regarding the existence of the glymphatic system , 141–143Of interest is that “glymphatic clearance” of the CNS is more active during sleep  while ly+ and CD4+ T cells, respectively. As outlined above, initiation of immune responses within the CNS parenchyma itself has not been readily observed. Instead, soluble antigens may be drained along the pathways that have been proposed by the aforementioned tracer studies for uptake and presentation within the CNS‐draining lymph nodes or, alternatively, CNS border‐associated APCs take up and present antigens locally or may potentially traffic from the CSF spaces to the lymph nodes.Many fundamental questions remain about how soluble antigens or APCs utilize potential efflux pathways for initiation of an adaptive immune response or the maintenance of peripheral tolerance to CNS autoantigens. In the periphery, antigen‐specific activation of lymphocytes may occur within the affected organ itself, along the pathways of antigen drainage or within the draining lymphoid tissue. Almost all tissues in the body contain strategically placed APCs capable of presenting antigens by MHC class I or II molecules to CD8Pioneering work by the group of Helen Cserr and Paul Knopf in the late 1980s and early 1990s, investigated systemic humoral immune responses in rats using CSF injections through an indwelling cannula of different foreign antigens, such as human serum albumin (HSA) or chicken ovalbumin, or autoantigens such as myelin basic protein (MBP). Low volume injections of either HSA or ovalbumin into the CSF resulted in an antigen‐specific IgG serum antibody response with antibody‐secreting cells identified in both superficial and deep cervical lymph nodes and the spleen , 148. InWhile it is generally now accepted that border‐associated macrophages such as perivascular macrophages , 154 andRecently, the choroid plexus and the dura mater have been implicated as important neuroimmune interfaces. However, a key question that remains is how these immune cell ramparts in the choroid plexus stroma and the dura mater could potentially communicate with the CNS border compartments or the CNS parenchyma proper that are separated from these sites by one or even two brain barriers. The choroid plexus has no known lymphatic vessel network and, thus, would not be an expected site for the initiation of a classical systemic adaptive immune response. Yet, one study has proposed that T cells injected into the lateral ventricle can migrate across the choroid plexus epithelium and undergo activation by APCs within the choroid plexus stroma . It willIt is also intriguing that immune cells in the dura mater are not found to be evenly distributed but rather observed to accumulate close to the dural venous sinuses , 40. ThiIn addition, the CSF‐draining perineural spaces have yet to be closely examined as potential sites of immunosurveillance. While peripheral nerve myeloid‐derived cells have recently been identified , 165, itOur concept where the brain barriers play a central role in maintaining CNS immune privilege and immune surveillance raises the question of how these barriers form during embryonic development allowing for the establishment of CNS immune privilege. Protein concentrations in fetal CSF are much higher when compared to the adult. While some authors suggested that this is due to the embryonic immaturity of the BBB and BCSFB, others have shown that the high CSF protein concentration is rather a result of transcellular transfer of plasma proteins across the embryonic choroid plexus epithelial cells (summarized in ). CombinCommunication between the brain barriers with a potential impact in adapting barrier functions has also been observed in the adult where in case of neuroinflammation when BBB properties fail, induction of tight junctions between astrocyte end‐feet was observed suggesting establishment of a novel glial BBB for immune cell and immune‐mediator entry into the CNS .An increasing number of studies have provided evidence for a role of BBB alterations established during development in causing psychotic disorders like schizophrenia. These observations converge with increasing evidence for a role of immune dysfunction in these diseases  pointingFurther elucidation of the role of the individual brain barriers and how they control the entry and exit of immune cells and soluble immune mediators in the CNS may lead to new therapies for patients with neuroinflammatory disorders focused on limiting the severity of clinical disability or preventing secondary progression. In this context, the development of imaging modalities allowing determination of the functional and/or inflammatory status of the different brain barriers by magnetic resonance imaging or positron emission tomography imaging in humans would significantly advance diagnosis and treatment of neuroinflammatory disorders. To this end, studies exploring the molecular signatures of the brain barriers during health and neuroinflammation will set the stage for future developments of barrier‐specific imaging modalities. In this context, the recent high profile studies that have suggested possible roles for immune cells at the choroid plexus and the dura mater will spark this research on both the preclinical and clinical levels to find potential new targets for diagnosis and treatment at these CNS border tissues.A better understanding of the compartmental nature of the CNS may also improve drug delivery for treatment of neurological conditions. It remains a significant challenge for systemically administered compounds to reach target cells in the CNS parenchyma as they must not only breach the BBB or BCSFB but also the additional barriers at the pia mater and/or glia limitans. Alternative strategies may emerge utilizing intrathecal or intranasal administration to target immune cell aggregates within the CSF compartment or to modulate lymphatic efflux.In conclusion, the orchestrated function of the different endothelial, epithelial, glial, and fibroblast‐derived brain barriers in zoning the CNS has not received sufficient attention. Integrating our growing mechanistic understanding of the function of these brain barriers in zoning the CNS into compartments with different accessibility to immune cells and immune mediators will be of fundamental importance to understand how these neuroimmunological interfaces shape CNS immunity and neuronal activity and thus ultimately CNS health and disease.The authors declare no conflict of interest.


Tumor heterogeneity is primarily responsible for treatment resistance and cancer relapses. Being critically important to address this issue, the timely evaluation of the appropriateness of therapeutic actions at the single‐cell level is still facing challenges. By using multi‐functionalized nano‐systems with the delivery vector composed of histone for plasmids loading, hyaluronic acid for tumor targeting, and a fusion peptide for C‐X‐C motif chemokine receptor 4  with in situ detection on biomarkers at the single‐cell level is realized. By efficient co‐delivery of the genome editing plasmid for CXCR4 knockout and molecular beacons for detection of upregulated mRNA biomarkers into CTCs in unprocessed whole blood, the therapeutic outcomes of genome editing at the single‐cell level can be in situ evaluated. The single‐cell analysis shows that CXCR4 in CTCs of cancer patients is efficiently downregulated, resulting in upregulated anticancer biomarkers such as p53 and p21. The study provides a facile strategy for in‐depth profiling of cancer cell responses to therapeutic actions at single‐cell resolution to evaluate the outcomes of treatments timely and conveniently. Using multi‐functionalized nano‐systems to co‐deliver the genome editing plasmid and molecular beacons, the therapeutic outcomes of genome editing on circulating tumor cells of cancer patients can be directly visualized. This study provides a facile strategy for in‐depth profiling of cancer cell responses to therapeutic actions at single‐cell resolution to evaluate the outcomes of treatments timely and conveniently. However, analysis of the primary tumor and metastatic lesions by multiple and repeated tissue biopsies is not clinically feasible. An alternative strategy, analysis of circulating tumor cells (CTCs) as a noninvasive liquid biopsy technique provides unique insights into tumor heterogeneity by studying molecular features of cancers at single‐cell resolution. Besides, CTCs play a crucial role in cancer metastasis. Single‐cell profiling of CTCs can unravel real‐time responses to cancer treatment and evaluate metastatic risk.As a fundamental property of cancers, cancer heterogeneity resulting from genetic, epigenetic, and/or phenotypic changes leads to different levels of sensitivity to cancer therapies and provides the fuel for the development of therapy resistance, which becomes a critical hurdle in cancer therapy.1, 2 However, the safety and efficacy are critical concerns of genome editing, especially for in vivo studies. Enjoying safety benefits, the ex vivo editing approach not only allows therapeutic actions but also provides a facile platform for exploring cancer gene therapy. Most commonly, ex vivo editing involves complicated procedures . As far as we know, ex vivo genome editing on CTCs in whole blood has never been reported.As it is well known, genomic changes play a curial role in tumorigenesis and neoplastic progression. Genome editing is a robust strategy for cancer treatment and cancer research.6 HoweThe purpose of this study is to develop a platform for ex vivo genome editing on CTCs with in situ evaluation of therapeutic efficiency at the single‐cell level to unravel real‐time responses to cancer treatments for personalized cancer therapy. By using a highly efficient delivery vector based on natural occurring biomacromolecules to specifically deliver the CRISPR‐Cas9 plasmid and molecular beacons to CTCs in peripheral blood of cancer patients, the therapeutic interventions of genome editing can be assessed in situ by detection of multiple mRNAs including mRNA of the targeting protein and other mRNAs playing important roles in cancer progression. The genome editing was directly carried out in CTCs in whole blood without isolation to mimic the in vivo process as well as to eliminate the unfavorable effects of cell isolation on CTC activity.As far as we know, the co‐delivery systems for simultaneous gene editing and mRNA probing in CTCs have never been reported. Our investigation provides a facile ex vivo strategy for evaluating the efficiency of therapeutic actions in a particular patient to provide accurate information for personalized therapy by using a few milliliters of peripheral blood containing CTCs. For example, the CXCR4/CXCL12 axis promotes tumor progression and metastasis by regulating the MAPK and PI3K‐AKT pathways. In addition, the activation of CXCR4 promotes epithelial‐to‐mesenchymal transition (EMT) and increases the secretion of matrix metalloproteinases (MMPs) that facilitate the process of invasion. Inhibition of CXCR4 by CXCR4 antagonists, CXCR4 siRNA, and CRISPR‐based genome editing is a promising therapy strategy in the treatment of a variety of cancers.In this study, C‐X‐C motif chemokine receptor 4 (CXCR4), a typical target protein in cancer therapy, was selected as a representative target of ex vivo genome editing. As a promising therapeutic target and an important prognostic cancer biomarker, CXCR4 plays a vital role in the crosstalk between cancer cells and the microenvironment for cancer development.12 For various non‐viral vectors have been developed for delivering genome editing systems to cancer cells. However, as far as we know, there are no reports on the vectors for delivery of genome editing systems to CTCs in whole blood. Besides, among diverse non‐viral vectors, such as cationic lipids, cationic synthetic polymers, inorganic nanocarriers , our biomacromolecule based vectors exhibit ideal biocompatibility, which minimizes the unfavorable effects, such as perturbing microRNA levels in treated cells, to ensure the accurate detection on therapeutic responses.Since viral vehicles suffer from susceptibility to mutagenesis, carcinogenesis, and immunogenicity,25 var2Figure  The negatively charged HA can target CD44 receptors overexpressed on cancer cells, enhancing the identification of neoplastic cells. In addition, the degradation of HA by hyaluronidase triggers a charge reversion promoting the endosomal escape. The fusion peptides, T22‐NLS can specifically combine with CXCR4 overexpressed on cancer cells to promote cellular uptake by the T22 sequence, and enhance nucleus translocation by the NLS sequence. With the combination of these functional components, the multifunctional delivery vector can specifically deliver the geneediting plasmid to cell nuclei to effectively mediate CXCR4 knockout. The downregulated CXCR4 in cancer cells results in inhibited growth, migration, and invasion with downregulated Bcl‐2, p‐ERK, MMP‐9, and MMP‐13 and upregulated p53, p21, and Bax. By co‐delivery of a molecular beacon with the genome editing plasmid, the upregulated mRNA of a particular protein  can be in situ probed , the resultant CRISPR‐Cas9 plasmid@histone/HA (P@HH) and CRISPR‐Cas9 plasmid@histone/peptide/HA (P@HPH) exhibit negative zeta potentials. All nanoparticles show satisfactory plasmid encapsulation efficiencies higher than 85%  and quantitated by flow cytometry Figure 2. The cFor noncancerous HEK293 cells, although T22‐NLS and/or HA decoration increases the cellular uptake slightly, no statistically significant difference exists between different delivery systems . Both CLSM and flow cytometry show the fluorescence intensity induced by hybridization of the molecular beacon with p53 mRNA or p21 mRNA in edited cancer cells greatly increases as compared with that in unedited cells Figure 5.The important characteristics of cancer metastasis and recurrence are the capabilities of migration and invasion. In this study, the effects of genome editing on the proteins involved in metastasis and invasion were studied. As revealed by Western blot assay, phosphorylated ERK (p‐ERK) for regulating cell motility, snail, vimentin, and twist for promoting EMT, as well as MMP‐9 and MMP‐13 for degrading extracellular matrix are greatly downregulated in edited cells after CXCR4 knockout. Besides, E‐cadherin for maintaining cell–cell interactions are upregulated , the edited cells treated with P@HPH exhibit dramatically suppressed migration and invasion, that is, the number of migrated cells decreases by ≈60% and the number of invaded cells decreases by more than 80% after CXCR knockout  and the control plasmid delivery system (CP@HPH) were directly added into 2 mL of unprocessed whole blood from cancer patients, respectively. After 12 h, to avoid the unfavorable effects due to the viscosity enhancement of the whole blood, CTCs were isolated from the whole blood and the CTCs on the filter membrane were further incubated in DMEM for 36 h Figure 6a. The cAfter genome editing, to detect the level of CXCR4 mRNA, the molecular beacon delivery system with the same targeting vector, MB1@HPH, was added to CTCs, followed by incubation for 4 h to allow the delivery of MB1 into CTCs for hybridization with CXCR4 mRNA. For comparison, CXCR4 protein on the surface of CTCs was labeled by the CXCR4 antibody. CLSM observation shows the edited CTC has a weaker fluorescence generated from the hybridization of MB1 and CXCR4 mRNA as compared with the unedited CTC Figure . The comFigure For the proteins upregulated after genome editing, the molecular beacons for their mRNA detection can be co‐loaded with the genome editing plasmid to realize in situ evaluation in the reprogrammed CTCs. Herein, the CRISPR plasmid/molecular beacon co‐delivery system was added to 2 mL of whole blood of patients for CXCR4 knockout and in situ probing of unregulated mRNA as detailed in Herein, the molecular beacons probing p53 mRNA and p21 mRNA were designed based on the sequences of wild‐type p53 and p21. It should be noted that mRNAs of non‐mutated and mutated p53 and p21 may co‐exist in the samples from patients. It is possible that MBs may hybridize with some types of mRNAs of mutated p53 and mutated p21. Whether the hybridization occurs or not is dependent on the position of the mutations, which may be different for different patients. Theoretically, if we identify the detailed position of the mutations for each particular patient, we can optimize the design of MB sequences to ensure that MBs only hybridize with wild‐type mRNAs of p53 and p21. This is an area requiring further study. The above results imply that our genome editing systems may prevent cancer progression and metastasis by effectively reprograming CTCs to reverse oncogenic properties.As detailed above, the multiple functional delivery systems can realize efficient genome editing and mRNA detection on CTCs from cancer patients. As well known, high expression levels of CXCR4 are predictive of poor prognosis in cancer patients.51 TheBy using the multiple functional CTC targeting delivery vectors we developed, both therapeutic and diagnostic agents can be delivered into living CTCs in peripheral blood efficiently to exert ex vivo anti‐cancer treatments with in situ detection on therapeutic responses of particular patients. Owing to the superiorities of noninvasive blood biopsies such as easy accessibility for frequent detections, our approach provides an avenue to conveniently tailor and optimize therapeutic strategies according to the genomic characterization of CTCs and the therapeutic responses at the single‐cell level for each patient during the anticancer therapy.3In summary, by using multiple functional CTC targeting delivery systems, we realize robust genome editing in CTCs in whole blood from cancer patients. More importantly, the therapeutic efficiency can be in situ evaluated to provide important information for personalized cancer therapy. Due to the enhanced cellular uptake mediated by T22 targeting CXCR4 and HA targeting CD44 in malignant cells, our delivery vector can target cancer cells as well as CTCs in whole blood to realize efficient genome editing and mRNA detection. The genome editing delivery system exerts anti‐cancer effects through downregulation of proteins promoting cancer progression and upregulation of anticancer proteins such as p53 and p21. The edited cancer cells exhibit inhibited proliferation and suppressed migration and invasion. The therapeutic efficiency of genome editing in the cancer cell line as well as in CTCs can be facilely probed using the same targeting delivery vector to deliver molecular beacons into the living cells. The downregulated mRNA (CXCR4 mRNA) can be detected by a molecular beacon delivery system after the genome editing, and the upregulated mRNA (p53 mRNA and p21 mRNA) can be probed in situ by plasmid/molecular beacon co‐delivery systems. Through ex vivo studies on living CTCs in peripheral blood at the single‐cell resolution, this study provides a powerful and new platform to study the therapeutic responses of particular anti‐cancer treatments in cancer patients to provide accurate and timely information for personalized cancer therapy.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.