Chatbots are applications that can conduct natural language conversations with users. In the medical field, chatbots have been developed and used to serve different purposes. They provide patients with timely information that can be critical in some scenarios, such as access to mental health resources. Since the development of the first chatbot, ELIZA, in the late 1960s, much effort has followed to produce chatbots for various health purposes developed in different ways.This study aimed to explore the technical aspects and development methodologies associated with chatbots used in the medical field to explain the best methods of development and support chatbot development researchers on their future work.We searched for relevant articles in 8 literature databases . We also performed forward and backward reference checking of the selected articles. Study selection was performed by one reviewer, and 50% of the selected studies were randomly checked by a second reviewer. A narrative approach was used for result synthesis. Chatbots were classified based on the different technical aspects of their development. The main chatbot components were identified in addition to the different techniques for implementing each module.The original search returned 2481 publications, of which we identified 45 studies that matched our inclusion and exclusion criteria. The most common language of communication between users and chatbots was English (n=23). We identified 4 main modules: text understanding module, dialog management module, database layer, and text generation module. The most common technique for developing text understanding and dialogue management is the pattern matching method . The most common text generation is fixed output (n=36). Very few studies relied on generating original output. Most studies kept a medical knowledge base to be used by the chatbot for different purposes throughout the conversations. A few studies kept conversation scripts and collected user data and previous conversations.Many chatbots have been developed for medical use, at an increasing rate. There is a recent, apparent shift in adopting machine learning–based approaches for developing chatbot systems. Further research can be conducted to link clinical outcomes to different chatbot development techniques and technical characteristics. Chatbots are systems that are capable of conversing with users in natural language in a way that simulates the interaction with a real human. The development of chatbots has captured the attention of researchers for a long time. Eliza  was one Since the early years of their development, people adopted different techniques in designing and developing chatbots. In recent years, with the increasing adoption of machine learning and artificial intelligence methods in different application domains, state-of-the-art methods in dialogue generation and dialogue management are increasingly using machine learning and deep learning methods -6.The medical field is an application area where chatbots are increasingly being adopted as a tool to facilitate access to information from the patient side and reduce the load on the clinician side. Many commercial chatbot applications that are available as web or mobile applications have been developed for interacting with patients. Some examples of health care chatbots are OneRemission1, which was developed to help cancer survivors; Babylon Health, which is a symptom checker; and Wysa , which iIt is important to know the current state of different methods and techniques that are being employed in developing chatbots in the medical domain for many reasons. Conducting such a survey will help researchers in the future identify the different methods that have been used and to build on the existing approaches to develop more intelligent chatbots that provide a more natural experience to the user. It is also important to see where the current state of chatbot development stands with respect to developing chatbots for other applications. Therefore, in this work, we conducted a scoping review of the available literature on chatbot development in the medical field and constructed and identified the main components involved in chatbot development, as well as a description of techniques used in developing each of the identified components. The main objective of this study was to explore technical aspects and development methodologies associated with chatbots used in the medical field to explain best methods of development and support chatbot development researchers in their future work.This study follows a scoping review methodology. Specifically, it follows the PRISMA extension of scoping reviews . In thisWe searched 8 databases  to collect studies relevant to the topic. For Google Scholar, we only used the first 100 results from each search string, as Google Scholar returns the most relevant results belonging to each search query first. The search was conducted between September 9, 2019 and September 13, 2019. For the forward reference list checking, we used the cited-by functionality of Google scholar. We also checked the reference list of the included studies to review the backward reference list.We used 2 different sets of search terms to search the databases. The search term set was decided based on the type of studies indexed by the database. For databases that mainly indexed studies in the medical field , we relied on keywords that are strictly related to the intervention . For databases with no specific application domain, we resorted to using keywords that are related to the medical domain  in addition to the intervention-related words. The search strategy used for searching the databases is presented in The purpose of this work was to review the technical aspects of developing text-based chatbots in the medical field. Therefore, for a study to be considered, it had to satisfy the following criteria: describe a chatbot application, the chatbot must be developed for a medical application , the input or output modality of the chatbot must be text, and the technical details of how the input is processed and output is produced must be mentioned. Studies that used a Wizard of Oz experiment design were excluded. In addition, some restrictions on the language of the study and publication type were enforced. Only studies that were published in English were included, and only peer-reviewed articles, conference papers, thesis, dissertations, and industrial and academic reports were considered.The study selection was conducted in 2 stages. Title and abstract screening was followed by a full-text screening stage. Both stages were conducted by 2 reviewers. The first reviewer, ZS, performed the screening of the full set of articles. Due to time constraints, the second reviewer, AA, reviewed a randomly selected set of 50% of the articles. Disagreements between the reviewers were resolved by a third reviewer, MH. To evaluate the interrater agreement, we used Cohen kappa . The revThe data extraction was conducted by ZS following a preset form. The data extracted pertained to the metadata of the included studies as well as the different technical modules of interest in the study, such as the text understanding module, text generation module, and method of linking these modules. The data extraction form is shown in As this is a scoping, not a systematic, review, no study quality assessment was conducted for the purposes of this work.We used a narrative approach to synthesize the different reported results. We included a description of the included studies and a description of the different techniques used for the development of the chatbots.The included studies were published between the years 2009 and 2019, with 80% (36/45) of the studies published in the last 5 years, in the years 2015-2019. Conference papers were the most common publication type , followed by journal papers  in addition to 2 magazine articles and 1 thesis. The most common country for publications was the United States, with 8 studies, followed by Australia, India, and Switzerland, with 4 studies each. The total number of chatbots assessed in this study was 45. The chatbots were developed for different diseases and to fulfill different purposes. An important factor for the technical aspects of developing chatbots is the language that the chatbot uses for communication. The majority of chatbots communicate in English , 4 in German, 3 in Chinese, 2 in Arabic, 2 in Korean, 1 in Thai, 1 in Spanish, and 1 in Russian; 8 studies did not mention the language. The chatbots operated as either standalone applications (n=17) or web applications (n=20), while the remaining studies did not mention the application type (n=8).The chatbots in the included studies consisted of 4 main modules: text understanding module, dialog manager, text generation module, and database layer that holds the various types of information needed for chatbot training and function. The text understanding module is the module with which the user directly interacts. The function of the text understanding module is to extract meaning from the user input before a specific answer is generated. In the included studies, 7 methods were used for text understanding. Pattern matching methods were the most common, used by 18 of the 45 chatbots (40%). Many text understanding approaches can fall under pattern matching. The different pattern matching methods reported in the included studies are as follows: keyword matching or string matching, where specific keywords or strings of the user input are matched to scripts stored in the database, which was used in 11 studies ,46,48,50In the reported studies, the input to the dialog management module is the processed user free-text input provided by the text understanding module. The dialog management module controls the different aspects of the conversation and links each user input to an appropriate output. In the included studies, 2 main types of dialog management techniques were reported: static dialog management and dynamic dialog management. In the case of static dialog management, user input is matched directly to the appropriate output using a pattern matching algorithm (25/45) or using a set of rules (7/45). In the case of dynamic dialogue management, the context of the conversation changes based on specific user input characteristics. The context switching can be done by training machine learning algorithms to identify the context from the user input (5/45) or using a web service for intent identification (4/45). Most of the chatbots in the included studies contained one or more of the 3 data repository types identified. Most of the included studies kept a medical knowledge repository . The medical knowledge repository contains medical information related to the application domain of the developed chatbot. The medical knowledge source can be local, such as obtaining it from medical personnel, or it can be collected from online sources such as Wikipedia or other websites. The medical knowledge can be presented to the user in the context of educational chatbots (17/45) ,48-51,53The text generation module provides output to the user. Text generation in the included studies was done using one of 2 methods: fixed output or generated output. The fixed output methods search the database for the most appropriate output to a user input and present it to the user. The generated output method relies on machine learning to generate original natural language output that is produced by the machine learning algorithm. Chatbots in most of the included studies provide fixed output that is extracted from the database with the exception of those in ,39,51, wA general architecture was identified and reported to summarize the technical aspects of chatbot development. The main components of chatbots, as well as the way these components are linked, are reported. Chatbots typically consist of 4 main components: text understanding module, dialogue management module, data management layer, and text generation module.The most common design method employed in developing chatbots is pattern matching for text understanding and response generation. Machine learning and generative methods are among the least commonly used methods for the development of chatbots in the medical domain. This can be attributed to 2 main reasons. The first reason for relying on pattern matching approaches more than those based on machine learning is that pattern matching methods are more reliable in practice because they produce exact responses to well-defined queries, resulting in fewer mistakes. Machine learning–based methods usually produce different types of errors, which cannot be tolerated in medical applications. The second reason for this trend is the rapid development in the state of the machine learning field over the past few years and the increase in the robustness of its methods, especially with the emergence of deep learning. While older methods relied on rule-based chatbots and pattern matching algorithms, all the proposed methods that rely on machine learning for text understanding and response generation were proposed between the years 2017 and 2019. Another reason for the possible lack of using machine learning methods could be the fact that machine learning–based approaches need to be trained using large amounts of domain-specific data, which might be scarce and difficult to access in the medical field. Overall, machine learning approaches and algorithms were better suited for developing chatbots used for specific medical conditions, such as mental health and autism, while the rule-based approaches were better suited for developing chatbots used for general medical purposes. On the other hand, pattern matching methods and algorithms were more broadly used in developing chatbots used for both special and general medical conditions.In terms of data management, the developed chatbots kept track of 3 different types of databases: medical knowledgebase including a library of medical facts, user information database including details about users’ demographics and their preferences, and dialogue script database including all possible entries of conversational text responding to users. The type of database kept depends on the chatbot type and target functionality. Educational chatbots usually keep a medical knowledgebase. Chatbots that use context switching based on user emotions usually keep a user information database.Most of the developed chatbots used English as the language of communication with the users, while other languages such as German, Chinese, and Arabic were less common. This is consistent with the fact that most of the publications originated in the United States, followed by Australia, where the first language is English.This review focused generally on chatbots in the medical field, without specifying the field of application, which makes it more comprehensive than previously conducted reviews . Other rThis review only focused on text-based chatbot applications, where either the input or output modality is written. This excludes studies where the input or output modalities are spoken or visual, as well as robotics and telephone-based methods. This choice was made because we wanted to focus on text processing techniques rather than image or voice processing, as speech-to-text technologies can also introduce errors and another layer of complexity to chatbot development.We enforced some constraints on the type of publications that were included in the current review. These constraints might have led to missing a portion of developed chatbots that have been published in other research venues, such as workshops, book chapters, and conference abstracts. Furthermore, limiting the search to papers published in English could also have led to missing some chatbots that were developed for communication in other languages and published in their own language. For example, we did not include papers published in Chinese or Arabic that discuss chatbots communicating in these languages.This review focused on the development process of chatbots without considering the impact of these methods on patients. For this reason, some of the implementations in some of the included studies might be feasible from a technical point of view, but this does not necessarily mean they are effective from a medical point of view.This paper reports the technical aspects of developing chatbots in the medical field. This review can be used to identify the most common development approaches by specialists to help them narrow down their options and make a decision on which development approach is the most appropriate for their applications.The reported results show that most of the developed chatbots communicate with users in the English language. While a few attempts to design and develop chatbots in other languages exist, more work needs to be done in this regard, especially for languages that are spoken by a large portion of the world’s population, such as Chinese and Arabic.Even though dynamic dialogue management provides a more natural user experience, most developed systems rely on static dialogue management methods. Changing the dialogue context based on user emotions or by detecting topic changes in user input are important aspects to be considered in chatbot development.The use of machine learning and artificial intelligence methods in the development of conversational agents in different application areas has recently increased. The rate of adoption of machine learning–based methods in developing chatbots is still relatively low, even though it has been increasing in recent years. Supervised machine learning algorithms seem to better suit the development of chatbots for special medical conditions and diseases, while rule-based methods are being used more for developing chatbots used for general medical purposes. Machine learning methods allow the development of more intelligent agents that can provide a more realistic user experience, by providing a better text understanding experience, including more dynamic and flexible dialogue management, and generating a wider range of responses.As the purpose of this review was to survey the technical aspects of chatbot development, the clinical results of performing clinical trials were not considered. Further reviews linking the different development techniques used to the clinical outcomes of the chatbot developed are possible and recommended.More openness and a wider adoption of state-of-the-art methods in dialogue management , text unIt is worth noting that the technical aspects of developing chatbots were not always clearly mentioned in the studies. The devised architecture is a general one that does not necessarily apply to every developed chatbot. One or more component might be omitted, and the chatbot might still function properly.In the scope of this review, we analyzed the technical aspects of developing 45 text-based chatbots for the purpose of performing different medical interventions. The most common language used for chatbot communication is English. Chatbots typically contain 4 main components: text understanding module, dialogue manager, database layer, and text generation module. The most common technique for developing chatbots is using a string matching algorithm and a set of scripts that contain sample inputs and outputs. Judging from the publication years of the different studies, we can conclude that chatbots are becoming increasingly popular for medical application, especially when it comes to mental health. The adoption of machine learning and artificial intelligence–based techniques has recently increased. Some development approaches are better suited than others for developing chatbots for specific medical conditions rather than general medical conditions. Future studies can be conducted to link the development techniques of chatbots to their clinical outcomes. It is important to conduct more in-depth systematic reviews on the effectiveness of chatbots in supporting and enhancing positive clinical outcomes. We need to understand and correlate different technical criteria and development methodologies to different levels of chatbots acceptance, utilization, and clinical effectiveness. Discussing the pros and cons of each chatbot system has also been left to future supplementary studies, to compare advantages and disadvantages of each chatbot system and link these to their postimplementation clinical outcomes.


Globally, an estimated two-thirds of all deaths occur in the community, the majority of which are not attended by a physician and remain unregistered. Identifying and registering these deaths in civil registration and vital statistics (CRVS) systems, and ascertaining the cause of death, is thus a critical challenge to ensure that policy benefits from reliable evidence on mortality levels and patterns in populations. In contrast to traditional processes for registration, death notification can be faster and more efficient at informing responsible government agencies about the event and at triggering a verbal autopsy for ascertaining cause of death. Thus, innovative approaches to death notification, tailored to suit the setting, can improve the availability and quality of information on community deaths in CRVS systems.Here, we present case studies in four countries  that were part of the initial phases of the Bloomberg Data for Health Initiative at the University of Melbourne, each of which faces unique challenges to community death registration. The approaches taken promote improved notification of community deaths through a combination of interventions, including integration with the health sector, using various notifying agents and methods, and the application of information and communication technologies. One key factor for success has been the smoothing of processes linking notification, registration and initiation of a verbal autopsy interview. The processes implemented champion more active notification systems in relation to the passive systems commonly in place in these countries.The case studies demonstrate the significant potential for improving death reporting through the implementation of notification practices tailored to a country’s specific circumstances, including geography, cultural factors, structure of the existing CRVS system, and available human, information and communication technology resources. Strategic deployment of some, or all, of these innovations can result in rapid improvements to death notification systems and should be trialled in other settings. Reliable evidence on the level and causes of the estimated two-thirds of global deaths that occur in the community is poor , with leDeath notification is usually performed by a designated agent or official, who generally provides the minimum essential information to initiate further government processes Table . ImportaWhile the complete registration of deaths with full accompanying information on the decedent should be the goal for any routine mortality surveillance system, notification of the fact of death, along with essential minimum demographic information, can serve several policy needs, particularly if the notification is linked to a procedure for a VA to ascertain probable COD. In such a system, all notified deaths would be registered, as is required for subsequent civil procedures and, in many cases, burial. This is the case, for example, in Kenya and South Africa, where notification and registration are linked and need to be completed in order to obtain a burial permit .In most low- to middle-income countries (LMICs) the increasing accessibility of information and communication technologies (ICT) can be harnessed to facilitate innovative methods for the notification of community deaths. In Uganda and Mozambique, for example, community notifiers use a mobile phone to send death (and birth) notifications to a central server, which is verified and registered by registration officers , 9. In NThere is, however, less evidence as to how death notification can be sustainably linked to local community structures, such as health clinics and aid posts, to ascertain the COD. Determining the underlying cause for all deaths is vital for a country’s public health planning and social development agenda. This is reflected in the United Nations Sustainable Development Goals, which have 17 indicators requiring COD data . A notifUnder the Bloomberg Philanthropies Data for Health (D4H) Initiative, 16 countries undertook a structured mapping exercise to identify and map current procedures, responsibilities and data flows for their CRVS systems . This reA conclusion from the mapping exercise was that countries required tailored approaches to strengthen and develop systems to suit their specific circumstances and available resources. To illustrate the innovative methods of improving death (and birth) notifications in resource-poor settings, we present case studies of four D4H partner countries: Bangladesh, Colombia, Myanmar and Papua New Guinea (PNG). The approaches taken demonstrate the ingenuity and flexibility required to address the complex challenges faced by these countries and highlight the significant potential that improvements to death notification systems can have to rapidly strengthen the evidence base of community deaths and their causes for policy and planning.Less than 10% of deaths are registered in a timely manner in Bangladesh, despite it being mandatory . A key rThe intervention involves community health workers using their routine household visits and regular ongoing Extended Programme on Immunisation services to identify deaths that occur in each local area under their mandate, assisting relatives of the deceased to complete the paper death registration application form. The form is then submitted to the health worker’s supervisor, who submits it to the local registry office where the ‘Union Parishad Chairman’ verifies the application, uploads the data into the online system and prepares the death certificate for the family. This linkage of notification and registration means the family only needs to take minimal action for a community death to be registered, compared with the passive registration system.In Kaliganj, from September 2015–August 2016 (before intervention) to September 2016–August 2017 (after intervention), the percentage of all deaths that were registered within 1 year increased from 24 to 94%  . A key bColombia has a relatively well functioning vital registration system . HoweverLed by the Ministry of Health and local health authorities, and supported by D4H, the system used mobile phone text messages for initial reports of a death and a cloud-based database to compile the data. The strategy engaged a variety of community members for notification, including community health workers, religious leaders, police officers and traditional birth attendants. The following reporting methods were employed: (1) a free short messenger service (SMS) message sent by community leaders from rural or ethnic communities, containing minimum essential information about the event; (2) police and civil registry offices conveying known deaths to local health authorities; (3) administrators of community cemeteries in rural areas recording and sending an SMS for each death; and (4) paper reporting forms for communities where the telephone signal coverage is poor. The SMS messages automatically populated an online platform and alerted the local health authority, community health coordinator and surveillance officers about the death.Following receipt of either the SMS or paper forms, a nurse coordinator or surveillance officer assigned the case to an auxiliary nurse or health promoter who validated the information and checked whether the death was already included in the national CRVS database. For each death, the health worker visited the family to conduct a VA interview using a tablet and uploaded the data to a cloud database.As of February 2019, 279 deaths from 2017 and 2018, which had not been previously included in the national mortality database , were identified in the 14 municipalities through the different reporting methods. Of these deaths, a VA had been performed for 149, with a COD determined for 98% of them. The newly identified deaths meant an 11% increase in reported mortality  in 2017, a 10% increase in 2018, and an overall increase in reported rural population mortality of 16 and 14% for 2017 and 2018, respectively.The ‘Colombia Rural Vital’ strategy has been effective for both the local health authorities and communities in better identifying the true level and pattern of mortality in these underserved populations, thus providing the evidence for government policies and programmes to focus attention and funding on appropriate interventions. The strategy has now been adopted as a part of the routine extramural activities of local hospitals within the 14 municipalities, and 2 of the participating local health authorities  have allocated resources to ensure the continuity of technical assistance in 2019.In Myanmar, it is estimated that the CRVS system registers about half of all deaths. There is an established health infrastructure in the country, the frontline of which is formed by basic health staff (BHS), including 30,000 health assistants, midwives, public health supervisors and lady health visitors that service the predominantly (70%) rural population . Only abFrom April 2017 to March 2019, a pilot death notification intervention implemented across 42 townships  led to a significant increase in the number of reported deaths when coupled with the introduction of VA. Following a death in the community, the village health administrator or volunteer informally notified the BHS of the event. The BHS used an electronic tablet to register the death and perform a VA at the home of the deceased. Basic notification information, including the most probable COD, was automatically sent from the tablet to a server based at the CSO. Data verification and analysis was then conducted by CSO staff.Importantly, as a result of the intervention, representative information on the causes of community deaths in Myanmar became available for the first time. Death registration in the 42 townships increased, on average, by 75% (from 38 to 66%) since 2016, with some townships increasing death registration from less than 5% to more than 80% , all with varying geographies and local government structures. These three provinces had already started to use the new electronic National Health Information System to capture deaths in health facilities but community deaths continued to be unrecorded. In each district, the underlying strategy was for an initial report of a death to be delivered to the health centre either electronically or as a paper-based form, then for the health centre to verify the data, complete an electronic notification form and conduct a VA, finally uploading the data to a central server for consolidation and review at the National Department of Health in Port Moresby.In three subdistrict areas of Milne Bay, known as Local Level Governments (LLGs), mobile phones were distributed to ward recorders to send an SMS with basic notification information on community deaths to their nearest health centre. To compare SMS to paper methods in the same district, paper forms were distributed to ward recorders for completion and delivery to the health centre in the remaining three rural LLGs in Milne Bay. In West New Britain, due to better transport links but poor telecom networks, paper forms were used, which were distributed to ward recorders in all LLGs. In Western Highlands, paper forms were distributed to village health volunteers (who act as notifying agents). Field coordinators employed at each site supported the notifying agents and, in areas where the literacy level of volunteers was low (such as the Western Highlands), facilitated the completion of paper forms and transfer of data from communities to health centres.Both the paper and SMS strategies involved the health centres arranging visits to the households following initial notification, health workers verifying the information, completing electronic notification forms and a VA, and uploading of the data to a central server for review at the National Department of Health. The strategies acknowledge the issue of data security of electronic transmission of information. The SMS of the initial report of the death only included limited information of name and ward of residence, and therefore had limited potential data security issues. The more detailed information on the electronic notification form collected by the local health centre was transmitted using secure ODK software. The importance of data security issues is also reflected by the new Civil Registration Bill and Regulations, currently being drafted, that will facilitate transmission of notifications via mobile phone or paper methods.Data collection took place between June 2018 and March 2019, and from a baseline of either no or very few out-of-facility deaths being notified in these areas, both paper and text methods demonstrated that death notification coverage could be increased to about one-third, on averageThe evidence from PNG, arguably one of the most difficult countries in which to implement death notification systems due to difficult terrain, poverty and other factors, suggests that a mixed system, involving different notifying agents and notification techniques , when combined with oversight and verification by the local health authorities, can significantly improve notification and COD information about community deaths.Notification of the fact of death is a critical first step in any vital registration system intended to provide the essential health intelligence for policy and planning. Notifications have languished globally, in part because of passive reporting procedures, but also because of a lack of innovation and research on potential cost-effective ways to capture information about deaths. The examples from countries reported in this paper demonstrate that active death notification systems adapted to country circumstances can lead to significant improvements in knowledge about community mortality and COD patterns using automated VA methods. Use of key notifiers with knowledge of their communities and integration of ICTs, where appropriate, can significantly aid this process. There has been a lack of focused methods for improving death notification, hampering efforts to improve registration completeness. Notification, for example, as a step in the overall registration process, is not described in detail in the Principles and Recommendations for Vital Statistics Systems from the United Nations Statistics Division, which focus instead on the designation and role of informants ; this isA key finding from the country examples reported here is that the move from a passive to an active notification system can have almost immediate benefits. In Bangladesh, strategic re-deployment of existing health staff highlights how targeted sensitisation and training programmes can effectively enhance notification from among existing community health workers. This has also been demonstrated in Myanmar, with the integration of the new notification system into existing health structures, particularly in rural areas, proving very successful. In some contexts, as seen in PNG where reporting of events is challenging, government services and agents may not always be able to identify and report deaths for the more remote areas, although the experience in PNG suggests that higher reporting of events can also be achieved through the use of a number of different reporting agents in these areas.The application of ICT in these countries has generally been efficient and resulted in an increase in death notifications. However, the potential for success of such technologies can be limited by the underlying organisational structures and overall CRVS processes, as was observed in the MOVE-IT pilot study in Tanzania . SMS-basInnovative strategies to improve notification systems for community deaths  to inform government decision-making needs to be an operational research priority in LMICs. The four country examples reported in this paper demonstrate that systems tailored to a country’s geography, existing CRVS system, and human and ICT resources can be effectively deployed and will generate improvements in mortality data almost immediately. Such improvements to death notification systems can, and should, be trialled in many other settings to improve country knowledge about mortality levels and patterns – an essential source of evidence for policy and monitoring of progress against health and development goals.


Plasmonic particles as gold nanorods have emerged as powerful contrast agents for critical applications as the photoacoustic imaging and photothermal ablation of cancer. However, their unique efficiency of photothermal conversion may turn into a practical disadvantage, and expose them to the risk of overheating and irreversible photodamage. Here, we outline the main ideas behind the technology of photoacoustic imaging and the use of relevant contrast agents, with a main focus on gold nanorods. We delve into the processes of premelting and reshaping of gold nanorods under illumination with optical pulses of a typical duration in the order of few ns, and we present different approaches to mitigate this issue. We undertake a retrospective classification of such approaches according to their underlying, often implicit, principles as: constraining the initial shape; or speeding up their thermal coupling to the environment by lowering their interfacial thermal resistance; or redistributing the input energy among more particles. We discuss advantages, disadvantages and contexts of practical interest where one solution may be more appropriate than the other. Plasmonic particles are emerging as a versatile solution for a broad variety of unmet needs at the crossroads of biomedical optics, sensing and imaging, owing to their unique efficiency to enhance, absorb and scatter light ,2,3. In PAI is emerging as a powerful solution to combine the best advantages of optical contrast and acoustic detection . The keyI is optical power density [W/mAt the core of PAI is a process of photoacoustic conversion that transforms an optical pulse into an acoustic signal . In turnk is thermal conductivity [W/(m·K))].Then, the process of photothermal conversion and heat propagation is governed by the general equation:Instead the process of thermoelastic conversion and acoustic propagation in a liquid follows the equally general equation:F is the optical fluence [J/mIn the standard case, where the processes of optical absorbance and thermoelastic expansion occur in the same medium, and the entire process is fast enough for thermal conductivity to be negligible, i.e., int-like , the phoHowever, in a more general situation, such as that of our interest, where the event of optical absorbance takes place within the gold nanorods, but the process of thermoelastic expansion develops in their aqueous medium ,16, therPAI has been implemented in many labs and several platforms are available on the market for preclinical as well as translational or clinical work. In general, photoacoustic systems can be divided into two general classes , i.e., tAs we have mentioned, the primary mechanism of contrast in PAI is optical in nature. More precisely, it is often assumed that photoacoustic images encode maps of optical absorbance. However, unfortunately, other factors concur to provide contrast, such as the heterogeneity of optical fluence and Grüneisen parameter or acoustic reflections, and their consideration is an active field of ongoing research ,25. HereThe use of PAI for the investigation of biological dyes is becoming a clinical tool of much topical interest. However, it is understood that the potential of this technology may readily extend to the broader realm of generic molecular imaging by the use of molecularly-targeted optical contrast agents. At variance with ultrasound imaging, where contrast agents need to be in the micron range of size  and so cThere exist many materials that are under investigation as exogenous contrast agents for molecular PAI, and can be divided into two general classes: inorganic materials, such as noble-metal nanoparticles and nanocarbon, and organic materials based on organic dyes, semiconducting polymer nanoparticles, etc. ,37,38,39 systems , melanin systems  or polyd systems ,47. Conv systems ,49, carb systems  or graph systems , and a b systems  that oft systems , for theHowever, before we conclude this section, we briefly mention another popular direction, i.e., the use of multifunctional contrast agents for photoacoustic or multimodal image-guided interventions, such as photothermal or combinatorial therapies. Also in this case, one can find the same dichotomy between inorganic ,58,59,60This mini review will focus on plasmonic particles, and in particular on gold nanorods. We have chosen the case of gold nanorods rather than other shapes that are being assessed as contrast agents for photoacoustic imaging, like nano-shells , cages ,69 or stV is particle volume and There are many reasons why gold nanorods have become so popular as contrast agents for optical imaging, and in particular for photoacoustic applications, as confirmed by the increasing trend of number of papers over the years see . One of ectively :6)α1,2≡α1,2≡ϵ emquation (λLPR=λp[ϵAnother key feature of gold nanorods is their ease of synthesis and modification. The existing protocols of electrochemical ,83, seedIt is important to note that this range overlaps the transparency window of biological tissue of minimal absorption and scattering of light. Besides the aspect ratio, there are several other factors determining the exact position of the localized plasmon resonance wavelength. The first additional factor is shape. A prototypical rod shape is close to a cylinder with hemispherical ends (s-cylinder  or cigarFrom a chemical point of view, tuning the aspect ratio and volume of gold nanorods can be achieved through the variation of the concentrations of the various reagents, and more usually the amounts of seeds and Ag ions, in a step-wise manner. However, the desired effect can be obtained by additional procedures as well. Recently, some of us suggested two useful protocols for fine tuning the localized plasmon resonance wavelength and volume of gold nanorods based on a combination of overgrowth  and etchIn particular, the most important targets of gold nanorods are the optical hyperthermia and PAI of cancer. Since both applications derive from a process of photothermal conversion, despite fundamental differences, the rate of optical absorbance and non-radiative decay are key indicators for the performance of these particles as optical contrast agents. Various theoretical ,99,100 aAnother advantage of gold nanorods is their ease of modification with functional coatings. For most biomedical applications, the gold surface should be derivatized with appropriate biomolecules, such as antibodies, DNA, Raman reporters, fluorescent tags, stealth polymers, etc. ,104. BecApart from these advantages, the work on gold nanorods also meets critical issues that still represent a major barrier against their translational penetration. In most cases, the development of contrast agents for PAI looks towards a clinical use in the human body. Without delving into the multitude of regulatory and ethical issues associated to the development of pharmaceutical compounds, we just mention that their assessment is conditional to a number of prerequisites covered under the acronym ADMET, i.e., Absorption, Distribution, Metabolism, Excretion and Toxicity. We have already anticipated that, in terms of adsorption and distribution, the scientific community has made great strides towards efficient delivery. Also the issue of toxicity has undergone extensive research with overall positive outcome ,120. At The other principal issue for the implementation of gold nanorods as optical contrast agents for PAI is their photoinstability. This problem will be the principal subject of this mini review, and, in interest , and eveIn the paragraphs that follow, we explain the origin of this phenomenon, the methods developed for a rigorous analysis of relevant thresholds, and present a roundup of possible concepts and tools proposed for its mitigation.In this section, we provide a brief overview over some of the concepts found in the literature to explain the photoinstability of gold nanorods as a process inducing their transformation into more spherical particles, and why, when and how it typically happens. Without pretence of completeness nor theoretical formalism, we provide those elementary notions that may be more useful to create the essential background and underpin the rationale behind the practical guidelines described in Even at ambient conditions , but morIn the case of irradiation with short optical pulses, gold nanorods tend to overheat and then to cool back down to ambient temperature through a process that entails multiple consecutive steps: the optical activation of plasmonic oscillations, Joule heating by electron-phonon coupling in a timescale below 10 ps, and then thermalization with the environment by phonon-phonon coupling in a timescale that depends on various interface features and lies in the order of hundreds of ps ,131,132.2/ns , the typ2/ns . As a co2/ns , as willparticle .The deformation of noble-metal particles does not require their temperature to reach the melting point of the respective bulk material ,137. Ind∘C , or PEGy∘C [∘C  failed tFrom a practical point of view, the description of the exact pathway that leads to the spheroidization of gold nanorods is not as critical to the purpose of this work. However, it is interesting to note that the underlying process remains a subject of ongoing research. The fact that sharper tips are simultaneously less stable and more mobile than flatter plains has led most authors to postulate a gradual relocation of material from the end caps to the side walls of gold nanorods ,138,142.r papers ,143. By r papers . The phyr papers  and, perFinally, we mention that other processes that have been described in the literature include explosive fragmentation ,146, subIn this section, we present a brief overview over some of the experimental protocols reported in the literature to define and quantify the threshold fluences FAs already mentioned in the previous section see , commercOne of the most popular and straightforward protocols is based on the comparison of the spectra of optical extinction of the gold nanorods before and after exposure to the laser beam for a given number of pulses at a given average fluence ,149,150.An alternative approach exploits a photoacustic probe in the place of the spectrophotometric analysis to detect the transformation of the contrast agent after an optical excitation ,152. Thr samples ,152.Another method that exploits the dependence of the photoacoustic response on the optical absorbance of the gold nanorods  is basedrity see B. When tFinally, we mention the possibility for a more qualitative and expedite approach based on the continuous monitoring of the photoacoustic signal over time or number of pulses at a certain fluence of interest ,156. In To conclude this section with a note of caution, we remark that the translational relevance of all these protocols and designs for phantoms may arouse controversy, because particles intended for use in vivo are likely to lose their colloidal stability at some point after systemic injection, for instance upon clusterization within endosomal vesicles ,157, whiIn view of its importance in typical application scenarios, different authors have undertaken the development of solutions to mitigate the issue of the photoinstability of gold nanorods. Here, we propose a broad classification of some of these methods chosen for their relevance and sorted according to general concepts based on our understanding of the underlying phenomena. While we are aware of the pitfalls hidden in a strict compartmentalization, our work intends to bring some order in a literature that is recent but already fragmentary, and to orient the experimentalist who wishes to consider the use of gold nanorods in PAI. In this sense, we associate each method with due references and an overview over relevant protocols for their practical implementation, and with a brief but multidimensional discussion of their pros and cons for biomedical applications.This strategy intends to increase the thermal stability and to slow down the course of reshaping of gold nanorods by inhibiting their process of pre-melting, whether they be exposed to an optical excitation or left on a hot plate, in an oven, etc. The most common approach to this effect consists in constraining the initial shape and frustrating the process of spheroidization by the addition of rigid or soft shells. Kennedy et al.  have desThe use of rigid shells is arguably the most popular and earliest solution implemented for the stabilization of the photoacoustic response from gold nanorods. In particular, the most common shell material is silica or organosilica, which enables fine control over parameters as thickness or the level of porosity . An enhaIn this context, the use of soft shells may be a beneficial alternative to brittle silica as well. Several authors have predicted  and obseFinally, we mention that a possible trade-off between the extremes of hard and soft shells may be metal shells, which hold the potential for a functional combination of stiffness and plasticity. In a recent work , we haveA common approach to enhance the functionality of gold nanorods is the formation of inorganic or polymeric shells. A first choice in this context is the addition of silica (general types of gold nanorod-based core/shell particles are shown in The protocols for the deposition of mesoporous silica often suffer from poor control over thickness. Other popular protocols to coat gold nanorods with silica derive from Stöber process in the presence of templating particles . Stöber The use of organic polymers is another popular option to coat gold nanorods. Usually, the polymerization of monomers on the particle surface is a substantial challenge that conflicts with the colloidal stability of the suspension. However, polydopamine has recently been suggested as a robust and convenient coating for various kinds of particles ,186. PolFinally, we also mention that gold nanorods can serve as a template for the synthesis of all-metal core/shell nanorods. The formation of uniform shells of silver on gold nanorods is a useful method to fabricate systems with new optical properties. One of the first reports on silver-coated gold nanorods with controllable shell thickness was published by Ah et al in 2011 . A modifIn conclusion, the scientific literature provides multiple examples of protocols to coat gold nanorods with hard, soft or other shells, such as silica, organic polymers as well as newer solutions as metals, which hold the potential to enhance their photostability. The use of silica or organosilica is the safest bet for its recurrence in the literature. But the other options may offer advantages in terms of ease of synthesis and purification.Coating with rigid or soft shells obviously implies a certain level of additional complexity that alters multiple features of the initial particles, including their hydrodynamic and electrokinetic profiles. Such modification may come with advantages and disadvantages but, in any case, needs understanding within an overall design of the system. Practical disadvantages include a probable complication of the procedures for purification, due to the multiplication of the steps of synthesis and a possible increase in the specific surface area of the particulate. For instance, gold nanorods modified with organosilica tend to retain a higher fraction of cytotoxic impurities used in their synthetic protocols , such asThe most obvious advantage of a strategy based on the enhancement of the thermal stability of gold nanorods is a simultaneous protection of both channels of photoacoustic as well as photothermal conversion , at variOverall, the addition of rigid of soft shells to mitigate the photoinstability of gold nanorods used as optical contrast agents for PAI is an effective approach that makes particular sense in a context where the the implementation of this solution belongs to a multidimensional rethink of the particles and their functions.Another critical factor is thermal coupling to the environment. We have already mentioned that thermal coupling between gold nanorods and their environment is a critical step in the photoacoustic process, because the photothermal conversion arises within the particles, but the thermoelastic expansion occurs in their dispersion medium. In practice this factor may be optimized either by lowering the thermal resistance at the interface between the particles and their dispersion medium or by increasing the specific surface area of the particulate.The notion to purposely minimize the interfacial thermal resistance is a difficult one, because a rational engineering of phonon-phonon coupling at a heterogeneous boundary remains a tremendous challenge. The thermal resistance at the interface between gold particles and their aqueous environment was shown to vary between about 2 and 30 × 10eat flux , nanoscaeat flux , radius eat flux  and wetteat flux . When exeat flux , such aseat flux ,163,164,eat flux ,220, itseat flux  reportedThe idea to rationally maximize the specific surface area of the particulate is more feasible. One possibility may be to miniaturize the gold nanorods. To a first approximation, according to Gans theory , dividinBack in 2014, some of us demonstrated a striking gain in photostability upon miniaturization of gold nanorods . A decreIn a recent work, we have proposed an alternative concept to increase the interface contact area between the gold nanorods and their aqueous environment by the deposition and galvanic etching of a silver shell conveying a rough or porous gold overcoating . We founIn the beginning, miniaturization has been pursued as a precondition to maximize the optical absorbance of gold nanorods per unit volume, and so their efficiency of photothermal conversion. The common seed-mediated synthesis gives particles with diameter larger than about 10 nm, and their cross sections for optical scattering are a minority but non-negligible fraction of those of optical extinction . The mosEl-Sayed and coworkers  developeprotocol . These a as 5 nm C. In linompounds  and by aunds [∘C  to improOverall, both strategies based on increasing the dose of seeds or directly injecting NaBHcibility . But theAs we have already mentioned in However, this said, there also exist very cogent arguments in favor of particle miniaturization. In principle, accelerating the thermal coupling between gold nanorods and their environment does not only affect their photostability, but may also enhance the efficiency of photoacoustic conversion, because the source of pressure associated to the thermoelastic conversion in the suspension medium scales as the square of the time derivative of temperature. This effect was invoked in the case of silanized  as well Overall, particle miniaturization or any other way to enhance their rate of dissipative cooling is an effective tool to gain photostability for PAI. However, this solution is less universal than the concept to improve their thermal stability, and probably requires careful verification from case to case. In any way, we believe that particle miniaturization may be the lifeline for a clinical exploitation of gold nanorods, should this roadmap really prove to convey biocompatibility. Therefore, photostability may become an important side benefit for the translational development of this material.Before we conclude this paragraph, we also point out that the concept of thermal coupling to the environment and its competition with a loss of thermal stability may arise also in contexts where an increase in specific surface area is not purposely targeted towards a gain in photostability. For instance, in a recent contribution , some ofIn previous sections, we believe that we have summarized the two most established highways to enhance the photostability of gold nanorods for PAI. Of course, there may also exist alternative or synergistic solutions. For instance, we suggest that another potential strategy may arise from the general notion to share the same extent of optical absorbance among more particles in the ensemble. The fact that the shape of gold nanorods is intrinsically anisotropic and typically inhomogeneous from particle to particle translates into a broad heterogeneity of photophysical behaviors. In practice, when monochromatic and linearly polarized light is used as optical trigger, energy is absorbed only by a small fraction of gold nanorods that enjoy near-resonant aspect ratio and proper alignment between their longitudinal axis and the electric field. In a recent publication , some ofAn equivalent variant to this idea to consider the effect of inhomogeneous broadening in the particle ensemble, may be to enforce a concept of homogeneous broadening, where a smoother plasmonic band in each particle would result into more particles resonating with less intensity over the same monochromatic excitation. This effect may for instance concur in the case of particle miniaturization, where smaller particles tend to exhibit broader peaks of optical absorbance ,231, andIn this article, we have highlighted different approaches to enhance the photostability of gold nanorods for applications in photoacoustic imaging. Despite the numerous advantages of gold nanorods associated with their tunable plasmonic resonances and extensive possibility of bioconjugation, their practical use as contrast agents in photoacoustics is limited by their ease to undergo photodamage. The photoinstability of gold nanorods is not only a phenomenon of academic interest, but also a concern of very practical relevance. Gold nanorods tend to lose efficiency of photoacoustic conversion upon exposure to ns-long pulses well before 10 mJ/cmWe have tried to classify the strategies used to increase the photostability of gold nanorods according to few basic principles. First, the use of rigid or soft shells is arguably the most popular solution implemented to constrain the initial shape and inhibit the progress of premetling. The use of silica or polymer coatings of a thickness in the order of several tens of nanometers increases the photodamage thresholds of gold nanorods by several folds. Another useful approach relates to lowering the thermal resistance at the interface between gold and water, which may be achieved by a decrease of average size or, for instance, an increase of roughness. In particular, particle miniaturization easily conveys an effect of an extent similar to that of silica coatings. Finally, we suggest that biggest advantages may be achieved by shifting the focus to the optical source, in order to redistribute the input energy among more particles by delivering non-polarized and non-monochromatic light.While more ideas are expected to unfold in the years to come, the combination of these solutions is on track to convey the due compliance with the conditions in use in preclinical and clinical systems for photoacoustic imaging. We are confident that our work will inspire new research towards a broader use of gold nanorods for applications in photoacoustics.


Background: There are concerns that the use of antibiotics before, during or immediately after pregnancy may have adverse effects on the neonatal gut microbiome and adversely affect the development of the infant immune system, leading to the development of childhood allergy, asthma, atopic disease and obesity.Methods: In this narrative review, we have explored a number of hypotheses, including the “Barker hypothesis”, the “hygiene hypothesis”, the link between inflammation and metabolic disease, and the influence of the neonatal gut microbiota on the development of the immune system in infants.Results: We found evidence to link the use of antibiotics before, during or immediately after pregnancy with an increased risk of childhood allergy, asthma, atopy and obesity.Conclusions: Although we found robust evidence to link antibiotic use in pregnancy with obesity and an “allergic triad” of asthma, eczema and hay fever, care must be taken when interpreting the findings because of the lack of adjustment for confounding variables in published studies. These may be (i) whether or not the mother had the same outcome variable  as the infant, for which the mother may have received the antibiotics; (ii) the indication, timing or number of antibiotic courses given; (iii) the use of broad-spectrum or narrow-range antibiotics; (iv) the dose-dependent nature of the effector; and (v) the class of antibiotics used. To establish a link between exposure to antimicrobials before, during and immediately after pregnancy with childhood inflammatory and metabolic diseases such as obesity, asthma and atopic disease, it is necessary to explore a number of hypotheses: (i) the Barker hypothesis, (ii) the hygiene hypothesis, (iii) the link between inflammation and metabolic disease and (iv) the influence of the neonatal gut microbiota on the development of the immune system in infants.1. The Romantic poet William Wordsworth, in his 1802 poem, “My Heart Leaps Up”, coined the idiom, “The Child is father of the Man” and this was used in the title of an editorial by Romero to illustrate how the Barker hypothesis pertains to perinatal medicine, which has major implications for obstetricians, neonatologists and society as a whole2. It is becoming increasingly more evident in several studies that insults beyond the obvious examples of genetic or congenital diseases during the earliest stages of life can profoundly affect our health as adults and cause disease. Intrauterine malnutrition is a significant risk factor for later stroke, diabetes, chronic hypertension and death from coronary artery disease in adults. Some causes of male infertility have their origins in inadvertent intrauterine exposure to environmental oestrogens. Under-nutrition during fetal or neonatal life may produce changes in haemostatic factors and in lipid metabolism, which increase the risk of cardiovascular disease. Raised cholesterol levels have been found in adults born with a small abdominal circumference, and increases in fibrinogen and factor VII are found in males born with short stature and a small abdominal circumference, who fail to gain weight during infancy. It is also possible that differences in diabetes, essential hypertension and other adult diseases observed in certain racial groups are not due to a genetic predisposition but are the result of differences in the quality of the intrauterine environment and postnatal life2. A large registry-based study of nearly 2 million Swedish children born between 1973 and 2014 reported the long-term risk of neuropsychiatric disease following exposure to infectionin utero. The exposure was hospitalisation during pregnancy with a maternal infection. The principal outcome measure was a diagnosis of psychosis, bipolar disorder, depression or autism among offspring. Although there was no evidence of a higher risk of bipolar disorder or psychosis among children exposed to infectionin utero, fetal exposure to a maternal infection raised the risk of an inpatient diagnosis of autism  or depression  in the child. Although the exposure was hospitalisation during pregnancy with a maternal infection rather than the use of antibioticsper se, the inference is that for an infection requiring hospital admission, antimicrobials would have been administered. These findings suggest that fetal exposure to a maternal infection during hospitalisation raised the risk of autism and depression. The findings emphasised how important it is to avoid infections during pregnancy, which may impart subtle fetal brain injuries that contribute to the development of depression and autism3.The “Barker hypothesis” – in which our health as adults and the quality of life of future generations may be determined by care or complications during fetal or early neonatal life, is now well recognised4. Although the terminology was not used, the message in this seminal publication has become known as the “hygiene hypothesis”4.In 1989, the results of a cohort of 17,414 British children born during one week in March 1958 and followed up to the age of 23 years were published. Of the 16 social, environmental and perinatal factors addressed, the most striking associations with hay fever  were those for family size and the position in the household during childhood. At the ages of both 11 and 23 years, hay fever was inversely related to the number of children in the household at age 11, an age when it was assumed that most families were complete. Eczema in the first year of life was also independently inversely related to the number of older children in the household. Strachan’s interpretation of the results was that allergic diseases were prevented by infections in early childhood, which were transmitted by unhygienic contact with older siblings or acquired antenatally from a mother infected by contact with her older children. Subsequent infection or reinfection by younger siblings might confer additional protection against allergic disease. Farm-raised children exposed to a variety of bacteria from animals and the soil also have lower rates of asthma. Strachan also hypothesised that, over the previous century, improvements in household amenities, declining family size, and higher standards of personal hygiene had reduced the opportunity for cross-infection in young families. He concluded, “This may have resulted in more widespread clinical expression of atopic disease, emerging earlier in wealthier people, as seems to have occurred for hay fever”5. The incidence of acute lymphoblastic leukaemia is rising by 1% each year in high-income countries and is more common in first-born infants. The theory behind this finding is based on a two-stage gene–environment interaction. The first stage occursin utero with an accidental gene mutation. The second stage occurs postnatally and makes the immune system more prone to react to infection, but only in children brought up in a relatively germ-free environment. According to Hawkes, “the first change loads the gun, the second pulls the trigger”5. In only-children raised in such a sanitised environment with little or no social contact in the first year of life, the development of the immune system is thought to be flawed. This flaw will be less evident in children who are breastfed and those born by vaginal birth. Accordingly, since the immune system evolves in an environment of infections, infection is required for it to form correctly6. Paradoxically, acute lymphoblastic leukaemia is caused by infections but also a lack of infection. The absence of “rough and tumble” in the first year of life leaves the immune system incapable of an appropriate response at a later stage. Although it could be argued that children today are exposed to infection through new vaccination programmes that stimulate the immune system, the antigens used to develop such vaccines are often based on attenuated viruses (as in varicella vaccine)7 or virulence factors such as capsular polysaccharides (as in group B haemolytic streptococcus vaccination)8 rather than the complete, wild organism that may be more antigenic and better able to stimulate an adequate immune response.In support of the hygiene hypothesis is recent evidence concerning the pathophysiology of acute lymphoblastic leukaemia. Acute lymphoblastic leukaemia is a disease of affluence and the commonest cause of childhood leukaemias. It is also the major cause of childhood cancer in high-income countries9. The acute response to inflammation is characterised by redness, fever, pain and swelling . This short-term adaptive response is a component of tissue repair and is advantageous to the organism. In contrast, the long-term consequences of prolonged, low-grade or chronic inflammation, that are metabolically triggered by nutrient and metabolic surplus, as in certain metabolic diseases, may not be beneficial and may predispose individuals to obesity and type 2 diabetes10.Among the most basic of defence responses to threat, whether in primitive organisms or in higher-order species, are the responses to a lack of nutrients (starvation) and the capacity to mount an effective immune response to pathogens (attack from a predator). Hence, many immune and metabolic response pathways, or pathogen-sensing and nutrient-sensing systems, alongside the molecules and pathways they comprise, have evolved in parallel and have been evolutionarily conserved throughout species12. This may be considered a form of “natural selection”. These traits in combination may generate an organism that is highly capable of processing and storing energy and is equipped with a powerful, and potentially hypersensitive, immune response. This relationship between the metabolic and immune response systems and the physiological mechanisms that control these responses in higher organisms has an evolutionary basis from common ancestry.The response to starvation selects for energy efficiency and favours the storage of excess calories when food is scarce. Conversely, with nutritional surplus, this once-advantageous metabolic state can lead to excess adiposity and its associated problems. The ability to resist infection has also led to Darwinian selection of a strong immune response. This is particularly evident when consideration is given to massive population declines during infectious epidemics and pandemicsEvolutionary perspective. An example of this is the fat body of the fruit fly (Drosophila melanogaster), which incorporates the mammalian homologues of adipose tissue and the haematopoietic and immune systems13 and has similar functional and developmental pathways9. Drosophila’s fat body coordinates the appropriate metabolic status and pathogen survival responses. In contrast, in higher organisms, the liver and haematopoietic system and adipose tissue have specialised into discrete functional units or organs but have maintained their developmental heritage, shared with more primitive organisms. Accordingly, the most primitive response systems integrate the pathogen- and nutrient-sensing pathways such that nutrients can induce immune responses and, conversely, pathogens can evoke and regulate metabolic responses.9. For the purpose of our review, we simply want to establish an association between inflammation and metabolic disease which provides a theoretical link between obesity and alterations to the development of the immune system.Taken together, inflammation and metabolic signalling create a delicate balance. Whereas the short-term compensatory and adaptive measures maintain this balance, when one arm overwhelms the other, the outcome is often detrimental in the long term. Accordingly, sustained exposure to pathogens can disrupt systemic metabolic function in both flies and humans. Similarly, chronic malnutrition or over-nutrition can disturb metabolic homeostasis and lead to an aberrant immune response. For those readers who wish to explore this theory further, linking inflammation with metabolic disease, we refer them to the excellent review by Hotamisligil14. Although the data are scarce, there also appears to be a rapid rise in childhood obesity in low-income and middle-income countries, despite continuing high levels of under-nutrition. Whereas data for children younger than 5 years have appeared in many large country surveys, data about older children and adolescents are less common, and sample sizes tend to be smaller. Nevertheless, comparable surveys show that the prevalence of overweight in these children is also rapidly increasing. A PubMed search indicates that, using the search term “childhood obesity prevention”, the number of published papers rose from about 20 per year in the late 1980s to 60 per year in the late 1990s and to more than 700 in 2018 alone15. At the time the manuscript of this review was submitted, there were already more than 600 such publications listed in 2019.In the past four decades, childhood overweight and obesity prevalence has risen substantially in most high-income countries16. A number of studies have addressed the use of antibiotics in pregnancy and subsequent childhood obesity21, only one of which found no association between antenatal antibiotics and childhood obesity21. A large Danish study indicated that school-age children up to 16 years of age, exposed to antenatal antibiotics, had a nearly 30% rise in the prevalence of obesity17. In another study, antibiotics administered in the second or third trimester, resulted in an 84% greater risk of obesity by 7 years of age when compared with those children not exposed to antenatal antibiotics19. Delivery by caesarean section (CS) compared with vaginal birth was associated with a 46% greater risk of obesity regardless of whether by emergency or elective section19. In a cohort of 527 children, antibiotics (particularly those given in the first and second trimester), but not antifungals, were associated with a raised body mass index by 2 years of age20. Although the weight of published evidence favours an association between the use of antibiotics in pregnancy and childhood obesity, we accept that obesity is a complex phenotype affectedinter alia by maternal nutrition and health before and during pregnancy. There were also many confounding factors, not fully addressed in the studies cited, such as maternal obesity and diet during pregnancy, gestational diabetes, gestational weight gain and the number of courses of antibiotics administered.With an important role in adipogenesis, any alteration in the gut microbiome increases the susceptibility to obesity in later life22, leading to long-term effects on immunological maturation and the potential for chronic disease23. In the human vaginal microbiota, in contrast to the human gut (where increased diversity is considered to indicate eubiosis), increased diversity is associated with dysbiosis, and low diversity of bacteria is considered to be an indication of eubiosis. Low diversity of bacteria has been found in the gut of neonates exposed to antibioticsin utero, particularly reduced levels ofLactobacilli andBifidobacteria, the primary organisms known to colonise the human gut in infancy and considered to be essential to initiate the immune system.The human gut is colonised by hundreds of microbial species that provide services (such as the production of enzymes and antitoxins that humans have not needed to evolve for themselves) but that also provide important functions, including development of the host immune system. The use of antibiotics in early infancy has been shown to cause intestinal dysbiosis25. In addition to having effects on the microbiome and metabolome, antibiotics may alter epigenetics26 and fetal development18; through these mechanisms, materno-fetal exposure to antibiotics increases the risk of immune-mediated diseases, such as asthma26.When used perinatally, antibiotics may disrupt the maternal-to-neonatal transmission of a healthy microbiota from the maternal gut, vagina, skin and breast milkLactobacilli27, and in babies exposed to antibioticsin utero, there is delayed colonisation of intestinal microbiota28. The gut microbiota profiles of 198 infants, whose mothers received intrapartum antibiotics, were associated with intestinal dysbiosis at the age of 1 year. This involved a reduction inBacteriodetes and greater quantities ofEnterococcus andClostridium species, frequently found in association with low gut microbiota diversity in patients with atopic disease. In one study, a degree of “microbiota recovery” throughout the first year of life was reported; this recovery was more evident in infants that were exclusively breastfed29.Prolonged rupture of the membranes and intrapartum antibiotics independently lead to a reduction in neonatal transmission ofin utero maternal microbiota and metabolites modified the fetal innate immune system and caused allergic airway disease31. The infant immune system is primedin utero and modified postnatally. Factors that modifyin utero microbial exposure may also influence the development of allergy32. Antimicrobials may induce a modification of the neonatal gut microbiome, which affects the developing immune system such that it promotes the development of allergic disease. Epidemiologically, the composition of the gut microbiota in children with atopic disease is different from that of normal controls33.To explain this observation, the biological model used involved the disruption of the developing gut microbiota, which resulted in a failure of maturation in the development of the neonatal immune response. This has also been demonstrated in animal studies, where the4, (ii) our adult health and the quality of life for future generations are determined by care or complications during fetal or early neonatal life1 and (iii) the development of our immune response from primitive life to the threat from infection is linked by the same molecules and pathways as our response to the threat of lack of nutrients9, it should not surprise us that the use of antimicrobials immediately before, during and soon after pregnancy may have long-term effects on the development of atopic and metabolic diseases such as asthma, eczema and obesity.If we accept the background developed from the “Barker hypothesis” section to the “Inflammation and metabolic disease” section that (i) the normal development of the immune system depends on antigenic exposure and our response to it34. However, antibiotic resistance remains a major problem35. The extent to which antimicrobials in general and antibiotics in particular are used in pregnancy is difficult to quantify as these may be administered in hospital, prescribed by primary care physicians in the community, or self-administered by pregnant women in the form of antifungals or antivirals purchased over the counter to treat self-diagnosed conditions such as herpes labialis, herpes genitalis or vulvovaginal candidiasis. Antimicrobials may be used for infections such as urinary tract infections, which are more common in pregnancy because of the physiological adaptations that are due to progestagenic effects on smooth muscle. Alternatively, they may be used for prophylaxis following preterm prelabour rupture of the membranes or to prevent early-onset neonatal group B streptococcal infection in women at risk of passing this on to the neonate or at the time of CS. A conservative estimate of 40% can be made from a Danish Registry–based study of nearly a million women36 but that is likely to be an underestimate as it applied only to antimicrobial prescriptions filled in the community without data on hospital-based or self-administered antimicrobials.Although concerns over teratogenesis from the use of antimicrobials in pregnancy are not major, the use of thalidomide casts a long shadow, so we should not be complacent37. The focus of the study was the risk ofClostridium difficile infection associated with the use of clindamycin and acute kidney injury associated with gentamicin during hospitalised birth. Although the absolute risk of such complications was low, the receipt of clindamycin was associated with significantly increased likelihood forC. difficile infection and receipt of gentamicin with significantly increased likelihood of acute kidney injury. Nevertheless, the impression given was that there was pressure for an increased, not decreased, use of antibiotics during hospitalisation37. This dichotomy may be emphasised by the recommendations for the early use of antibiotics when sepsis is suspected38.In a recently published study of 5.6 million pregnancies in the US between 2006 and 2015, 57% of women received antibiotics during hospitalisation for delivery39. The following year, it was noted that the prevalence of asthma and childhood eczema  had been increasing over the previous 30 years. Currently, atopic disease, a syndrome of hypersensitivity which typically presents as one or more of an “allergic triad” of asthma, eczema and hay fever, affects around 25% of the population, particularly in industrialised countries and especially in children and young adults40. The use of antenatal or intrapartum antibiotics is significantly associated with atopic dermatitis (eczema) in infancy if they are used for more than 24 hours41 or if the infant was delivered by CS42. In the first year of life, cow’s milk allergy (lactose intolerance) accounts for up to 6% of food allergies43. Food intolerance is probably due to a mixture of genetic and environmental factors but may be due to modifiable risk factors such as the use of antibiotics in pregnancy. Two studies reported that the use of antibiotics before or during pregnancy led to an increased risk of lactose intolerance in offspring, although this was less in children who were breastfed for longer periods of time45.In 1988, following successive reports from British General Practice Surveys, hay fever was described as a “post–Industrial Revolution epidemic”47. Asthma is also the most common chronic disease in children between 5 and 14 years of age46. Among the risk factors for asthma in offspring are maternal asthma, exposure to cigarette smoke, and air pollution in pregnancy49, together with low birth weight and preterm birth50. Even after controlled for confounding factors, an increasing number of studies have concluded that there is a positive association between childhood asthma and the use of antibiotics in or around pregnancy56. Vitamin D (the “antibiotic vitamin”) is an immunomodulator that is associated with a number of outcomes in pregnancy57 and extra-skeletal outcomes in children58. Supplementation with vitamin D in pregnancy reduces the risk of infection and the need to use antibiotics in pregnancy and also reduces recurrent wheeze and asthma in offspring in childhood at the age of three years59 though not at the age of six. However, care should be taken while interpreting such data. Two large registry-based studies56 suggested that the association between maternal antibiotic use and asthma might be due to a maternal propensity for infection rather than the use of antibioticsper se. The association was also stronger when antibiotics were prescribed for respiratory infections. A large population-based cohort study of mother–child pairs60 supported the contention that the association between maternal antibiotic use and asthma was not unique to use during pregnancy. However, it did provide evidence that maternal antibiotic exposure in a dose-dependent manner was associated with an increase in the risk of childhood asthma. After adjusting for infant gender, mode of delivery and method of infant feeding, most classes of antibiotics were associated with an increased risk of asthma, although the degree of risk varied.Over the last four decades, the global incidence of asthma has increased; it is estimated that there were 334 million cases in 2014https://food.ku.dk/english/research_at_food/research-projects/2014/neomune/.The Neomune Project was developed by the University of Copenhagen to study the role of diet and early microbial colonisation in the development of the neonatal immune system and cognitive function in vulnerable preterm or small-for-gestational-age infants. One aim of the Neomune Project is to investigate dietary and microbial elements that help maturation of the immune system in order to develop formula milk to support infants for whom breastfeeding cannot occur or is contraindicated. Another aim is to supplement the limited research on the use of probiotics in milk products. It is thought that these probiotic supplements have the potential to support the neonatal gut microbiome and to improve development of the immune system while suppressing pathogens in vulnerable neonates. For more information, see61. The evidence to provide confidence that vaginal seeding is safe in humans is not robust62, and there is a risk of neonatal transfer of asymptomatic vaginal pathogens such as herpes simplex virus,Neisseria gonorrhoeae andChlamydia trachomatis. Since intrapartum antibiotic chemoprophylaxis for the prevention of early-onset group B streptococcal infection of the neonate in the UK and many other European countries is risk-based rather than screening-based8 (as occurs in the USA), there are concerns that this significant cause of neonatal sepsis could be inadvertently transferred to the neonate by vaginal seeding. In the UK and Denmark, obstetricians advise against vaginal seeding and are required to inform women of the potential risks if they intend to pursue this route independently. The introduction of probiotics and breastfeeding are promoted by neonatologists. Ideally, new information about the species-specific function ofLactobacilli from the vaginal microbiome project63 will yield additional data for the development of improved probiotics65.First proposed in 2016, vaginal seeding (sometimes referred to as microbirthing) involves the postnatal transfer of maternal vaginal fluid to neonates born by CS, to correct the disruption of the neonatal microbiota that occurs with CS compared with vaginal birth66. Species ofBifidobacteria are dominant in human breast milk, protect the infant gut from pathogens, and provide health benefits later in life. However, numbers of these bacteria are reduced in the breast milk of women who deliver preterm and in those born by CS, although whether this relates to prophylactic antibiotic cover is unclear67. Accordingly, the American Academy of Pediatrics and the Royal College of Paediatrics and Child Health highlight the importance of breastfeeding and advocate the use of donor breast milk where maternal feeding is contraindicated, or not possible, rather than resorting to the use of formula feeds68.As the first type of food that is introduced into the infant gut neonatally, breast milk is thought to modify the developing microbiota, and, in babies breastfed within the first hour of life, there are similarities between the bacterial constituents of maternal colostrum and new-born meconium69. Although probiotics purport to balance the gut flora, reliable information is lacking with respect to materno-fetal and neonatal safety. In neonatal mice, T-cell disturbance secondary to the use of antibiotics was prevented by probiotics70, and improved mucosal tolerance following probiotics was reported71. Probiotics may reduce the incidence of pulmonary tuberculosis as well as allergies and asthma in children. However, a higher level ofBifidobacteria in infants given probiotics lasted only one week and was associated with an increased risk of infections in later life72. Accordingly, further well-conducted research is required; this will be covered as part of a separate review. Although increased diversity of the gut microbiota is considered a beneficial attribute, the opposite is true of the vaginal microbiota. New information about the vaginal microbiome is providing data about species-specific function of eubiotic, lactic acid–producing organisms that may lead to better probiotic therapies63, but the microbiome of the gut and the vagina are sufficiently complex that the use of a single probiotic, or even two probiotics, may not be able to address this complexity.The World Health Organization defines probiotics as “live organisms, which when administered in adequate amounts, confer a health benefit on the host”35 and (ii) the early use of intravenous antibiotics to reduce the serious morbidity and mortality associated with severe sepsis73.Increasing evidence links the use of antibiotics in pregnancy to subsequent childhood obesity, atopic disease and asthma. This effect appears to be long-lasting and so may also apply to pre-pregnancy and postnatal antibiotic use. Vitamin D, breastfeeding and vaginal birth may have protective effects. Interventions can include alterations to infant diet, breastfeeding or use of probiotics (or a combination of these). However, before “vaginal seeding” can be fully supported, more research is needed. Future research should include long-term follow-up and must be more robust with respect to correcting for confounding variables. It is uncertain whether the mother had the same purported outcome variable as the infant, such as asthma, for which the mother may have received antibiotics. In many studies, there is also a lack of control for the number of antibiotic courses administered, the timing of antibiotic use, whether broad-spectrum or narrow-range antibiotics were used, the indication for antibiotic use and the class of antibiotics used. Greater effort should be made to reduce the use of antibiotics in pregnancy. However, obstetricians are faced with the dichotomy of (i) pressure to reduce the use of antibiotics because of the problems of resistance