In 2013, the SickKids-Caribbean Initiative (SCI) was formalised among The Hospital for Sick Children in Toronto, Canada, the University of the West Indies, and Ministries of Health in six Caribbean countries . The aim was to improve the outcomes and quality of life of children (<18 years) with cancer and blood disorders in the partner countries. Core activities included filling a human resource gap by training paediatric haematologists/oncologists and specialised registered nurses; improving capacity to diagnose and treat diverse haematology/oncology cases; developing and maintaining paediatric oncology databases; creating ongoing advocacy activities with international agencies, decision makers, and civil society; and establishing an integrated administration, management, and funding structure. We describe core program components, successes, and challenges to inform others seeking to improve health service delivery in a multidisciplinary and complex partnership. In high-income countries (HICs), improvements in prognosis over the past decades have been notable, but have not been observed comparably worldwide.,,Latin America and the Caribbean have acknowledged health systems inequities, and cancer presents a growing problem.To address the observed inequalities in childhood cancer and blood disorders worldwide, twinning partnerships, specialised education, and telemedicine are promising, especially when situated within health system-wide efforts to improve care. These can offer healthcare professionals in resource-constrained settings improved access to focussed and continued training, and support in developing treatment and care plans, via the guidance and support of experts in resource-abundant settings.In 2013, healthcare professionals at a tertiary-care paediatric hospital in Toronto, Canada formally partnered with seven Caribbean institutions across six countries , with the primary goal of improving the outcomes and quality of life of children <18 years with cancer and blood disorders. This report outlines the core program components and identifies major successes and challenges, to inform others aiming to develop a multidisciplinary, complex partnership to improve health service delivery and patient outcomes.ad hoc physician-to-physician consultations on complex cases involving Caribbean children <18 years, the collaboration was facilitated by formal and informal relationships between physicians at the Hospital for Sick Children (SickKids) and the English-speaking Caribbean countries.SCI emerged in recognition of the need for improved paediatric haematology/oncology expertise and diagnostic service capacity in the partner six Caribbean countries. Inspired by ,In 2013, SCI was formally launched as a partnership among the Centre for Global Child Health at SickKids, the University of the West Indies (The UWI), Ministries of Health, and hospitals at seven sites in the six Caribbean countries. From the beginning, clear objectives and governance have grounded SCI, as further described by Manley-Kucey et al.Following phase 2, SCI has transitioned into a phase of long-terms sustainability underpinned by the establishment of an endowment by SickKids Foundation. Caribbean-based leadership has been further engaged, with SickKids continuing to provide administrative structure, access to case consultations and training fellowships, and some support for the Caribbean-administered paediatric oncology databases.•Physician-to-physician case consultations for complex paediatric haematology/oncology cases•Specialised education and training of physicians and nurses in paediatric haematology/oncology and SCD, including newborn screening•,Development of local paediatric oncology databases, including retrospective and prospective data collection, to reliably monitor incidence, treatments, and outcomes•Advocacy efforts and stakeholder engagement to raise awareness of the burden of paediatric haematology/oncology in the Caribbean, and the opportunity for outcome improvement.Several activities within SCI  aimed to,Underpinning all activities was a multi-level governance model, including administration, management, and funding support to ensure operational flow.Within the SCI partnership, four cross-cutting themes emerged: the formation of communities, in lieu of practicing in isolation; building local capacity, as opposed to ‘parachuting in’ with pre-identified priorities; a transition from supporting  to strengthening  the health system; and efforts to optimise practices in a setting-appropriate manner, rather than maintaining the status quo. These themes were identified internally by reviewing past internal and external SCI reports, and were approved by the SCI Research, Policy, and Advocacy Working Group. The following discussion of these themes highlights some of the key successes and challenges.The formation of formal and informal communities was an important consequence of SCI. As noted, prior to the partnership, three paediatric haematologists/oncologists practiced in the partner countries. Through SCI fellowships, this has increased to seven; 41 nurses have also received specialist paediatric haematology/oncology training.Building local ownership and initiative was a priority within SCI, and the sustainability of the project has been a core component of its mandate. To support the agency and development of the Caribbean-based healthcare professionals, establishing trust and open communication early on was critical. While this took time, including individuals from both the Caribbean and Canada in the governance structure and decision-making was felt to be critical to success, as were yearly in-person group priority setting exercises. Routine communications  also helped to maintain engagement and ensure that partners and stakeholders were aware of the status of the diverse activities underway. Given that the resources in the partner countries differed substantially from those accessible in HICs, ensuring that diagnostic and treatment plans aligned with setting-based realities were very important. Of the different core activities, the locally-built and initiative-informing databases, development of clinical and supportive care guidance documents, and strengthening of local educators and preceptors are examples of how local capacity was supported and facilitated.Within health systems, resources and infrastructure must first be in place before increased attention is focussed on strengthening efforts.An integral component of SCI was reviewing routine practices and identifying potential changes to improve patient outcomes and better fit setting-relevant contextual factors. Informed by case presentations and expert opinions, the development of locally-contextualised best practice clinical and supportive care guidance documents was one way this was achieved. The clinical care guidance documents were specific to different types of childhood cancer, outlining treatment recommendations, whereas the supportive care guidance documents focussed on preventing/managing complications due to cancer or treatments. Both guidance types were tailored to the unique resource limitations in the Caribbean context. Similarly, the incidence, treatment, and outcome data collected within the local oncology databases were considered with demographic and disease characteristics, to further understand and inform continuous improvement to clinical and supportive care. Reflection on practices and outcomes, and collecting corresponding data led to implementing meaningful changes.Successful partnerships between countries with different resource levels can serve as a springboard for systematic improvement to local patient care delivery. Prior to SCI, paediatric haematology/oncology was underserved in the English-speaking Caribbean, but through collaboration there have been significant improvements in the partner countries. Key outcomes of the SCI partnership have included increasing the number of trained local physicians and nursesSeveral factors facilitated the successes observed within SCI. Importantly, SCI was structured on engaging ethically and equitably to improve health. Priorities were co-developed, and respect was shown towards local culture and values. All partners, local and international, had a shared vision and goal: the advancement of paediatric haematology/oncology care in the partner Caribbean countries through capacity building; accurate and timely diagnoses; dedicated entry and tracking of cases in local databases; and development of critical human resources. Mutual trust and camaraderie underpinned the solid foundation between the Canadian and Caribbean partners. Reflecting on the Canadian Coalition of Global Health Research Principles for Global Health Research,Given the unique characteristics of each SCI partner country, developing a single model of care that worked for all six countries was unlikely. Barriers included differences in locally available financial support, population size, access to expertise, laboratory services, pharmaceutical/drug access, and governmental regulations. This led to variations in timelines and feasible accomplishments across the participating countries. For example, while there have been major strides in the care and outcomes for children with cancer and blood disorders in some of the larger SCI countries , the smaller countries have not experienced corresponding magnitudes of improvements . This can be largely attributed to resource constraints, both on the financial and supply sides. Generally, efforts to strengthen local infrastructure via governments have been slow, although there have been clear, significant gains through SCI.Interventions related to nursing focussed primarily on strengthening clinical competence through the development and implementation of a post-registration diploma in paediatric haematology/oncology nursing, as well as continuing professional development sessions.Regarding infrastructure, access to reliable radiological and pathological services and medical supplies was inconsistent. Considering SCD, for example, personnel training at one site to use heel prick and specialised high performance liquid chromatography testing was difficult, given limited local buy-in at the hospital- and/or Ministry of Health-level. In other cases, the paucity of local infrastructure presented a large enough barrier that access to necessary resources  were provided through SickKids. This is not sustainable, and efforts to transform these temporary supports into enduring and locally sustainable clinical programs are a crucial part of ensuring health system capabilities over the long-term. This highlights the ongoing struggles and need for continued advocacy and stakeholder engagement, as resolution of these challenges awaits further effort.The direct and indirect impact of SCI in the partner countries has been far-reaching. On the direct side, the increase in numbers of certified paediatric haematologists/oncologists has been a major achievement. The data collected within SCI has also led to novel clinical findings. For example, children with acute myeloid leukaemia (AML) were recognised as having poor outcomes across the SCI sites. An AML Working Group was therefore established to facilitate early discussion of newly diagnosed AML patients, between the managing local team and local and international experts. Although formal analyses are ongoing, initial findings suggests a trend towards a decrease in therapy-related mortality rates since these efforts were initiated. In highlighting the predicament of children with cancer and blood disorders in the region, a strong advocacy platform at the local, regional, and international level has also been established, including collaborations with the American Society of Haematology and the World Federation of Haemophilia.With the completion of phase 2 in March 2022, SCI has shifted towards a regionally led stage of sustainability. This has meant the decentralization of management from Canada to the SCI Caribbean sites, with some shared administrative responsibilities and ongoing case management support. To maintain the vision of SCI, empower local governance, and impact other countries throughout the Caribbean, the emphasis will be on creating a Caribbean paediatric haematology/oncology specialist-led culture of excellence. Achieving this will require dedicated local funding, coupled with continuation and enhancement of the SCI-based successes, such as data analysis, evaluation, strategic planning, engagement with Ministries of Health and regional healthcare agencies, and defined partnerships with international agencies and societies.To have the greatest impact possible for children <18 years with cancer and blood disorders in the region, leaders in the Caribbean will need to overcome site-specific differences and pursue a unified solution to existing challenges. Three top priorities going forward include (1) engaging Ministries of Health and governmental bodies to improve regional access to resources  and care; (2) continuing the paediatric haematology/oncology fellowship program to align the number of specialist physicians with local caseloads; and (3) use the local paediatric oncology data to continuously inform and improve treatment. Given the complexity of some paediatric cancer and blood disorder clinical presentations, ongoing case consultations with SickKids experts will be important. To enhance the gains of SCI, local consideration should also be given to support the training of key allied health professionals, including pharmacists, radiation therapists, and surgical oncologists.Through consistent efforts and dedication to the improvement of the lives of Caribbean children with cancer and blood disorders, the promise with which SCI started has, in large part, been realised. The initiative will continue to mature, helping these children to realize their full potential.MRM, CB, and JBB wrote the original draft of the manuscript, with input from VB. All co-authors provided input on the key activities and themes. All other  authors reviewed and edited the original draft, and approved the final draft.The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations.The authors  declare no competing interests.


Odoribacter, Allobaculum, Coprobacillus, and Alloprevotella were the dominant genera of the core fecal microbiota of coyotes. While for wild hogs, the fecal microbiota was dominated by bacterial members of the phyla Bacteroidetes, Spirochaetes, Firmicutes, and Proteobacteria. Five genera, Treponema, Prevotella, Alloprevotella, Vampirovibrio, and Sphaerochaeta, constitute the most abundant genera of the core microbiota of wild hogs in this study. Functional profile of the microbiota of coyotes and wild hogs identified 13 and 17 human-related diseases that were statistically associated with the fecal microbiota, respectively (p < 0.05). Our study is a unique investigation of the microbiota using free-living wildlife in the Texas Panhandle and contributes to awareness of the role played by gastrointestinal microbiota of wild canids and hogs in infectious disease reservoir and transmission risk. This report will contribute to the lacking information on coyote and wild hog microbial communities by providing insights into their composition and ecology which may likely be different from those of captive species or domesticated animals. This study will contribute to baseline knowledge for future studies on wildlife gut microbiomes.The ecology of infectious diseases involves wildlife, yet the wildlife interface is often neglected and understudied. Pathogens related to infectious diseases are often maintained within wildlife populations and can spread to livestock and humans. In this study, we explored the fecal microbiome of coyotes and wild hogs in the Texas panhandle using polymerase chain reactions and 16S sequencing methods. The fecal microbiota of coyotes was dominated by members of the phyla Bacteroidetes, Firmicutes, and Proteobacteria. At the genus taxonomic level,  Canis latrans) and wild hogs (Sus scrofa), two species of ecological and economic importance in the Texas panhandle. Biodiversity in the environment and wildlife is a key factor affecting the spread of infectious diseases. The majority of zoonotic pathogens such as Brucella spp., Toxoplasma gondii, pathogenic Escherichia coli, and influenza virus that can be transmitted to humans are transmitted by vertebrate animals, including feral swine and coyotes 30]. The  in pigs . On the llulitis ,33. Highpatients . Similarpatients . Oral anfections . Excessiaginosis . Treponeof swine ,39. Trepof swine . We founof swine , who expof swine .We also explored the functional profiles of the microbiota of both wild hogs and coyotes using the publicly available web-based MicrobiomeAnalyst in an attempt to gain more knowledge into the important functionalities of the gut microbiota of the wildlife species observed in this study. Some of the microbiotas of the wild hogs and coyotes have been associated with certain human-related diseases. We found an increased risk of susceptibility to Malaria, type I diabetics, Resistance to PD-L1 monoclonal antibodies, depression, Urogenital schistosomiasis, Colorectal Cancer, and resistance to an immune checkpoint inhibitor consistent with previous reports ,44,45. COdoribacter, Allobaculum, Coprobacillus, and Alloprevotella) also constitute the most abundant genera of the core microbiota of coyotes. The information provided in this study may be important in wildlife conservation, wildlife adaptation, fecal microbiota transplantation, and gut dysbiosis due to wildlife diseases and may also serve as baseline information for future studies, as well as comparative microbiota studies with domestic canids and swine. This report will contribute to the lacking information on coyote and wild hog microbial communities by providing insights into their composition, structure, and ecology which may likely be different from those of captive species or domesticated animals.In conclusion, we described the fecal microbiota of free-roaming coyotes and wild hogs in West Texas. The fecal microbiota provides information on diverse and abundant microbial communities that were unculturable and indescribable by culture-dependent methods. We detected dominant phyla  across the microbiome of coyotes and wild hogs. The four dominant genera identified (


To achieve the highest ethical standards in laboratory research, animal discomfort and the risk of health complications at the identification stage should be minimized. This requires the development of new bioethical yet reliable identification methods or the use of a combination of established non-invasive methods.Reliable methods for identifying rodents play an important role in ensuring the success of preclinical studies. However, animal identification remains a trivial laboratory routine that is not often discussed, despite the fact that more than 6 million rodents are used in animal studies each year. Currently, there are extensive regulations in place to ensure adequate anesthesia and to reduce animal suffering during experiments. At the same time, not enough attention is paid to the comfort of rodents during routine identification procedures, which can be painful and cause some complications. In order to achieve the highest ethical standards in laboratory research, we must minimize animal discomfort during the identification phase. In this article, we discuss traumatic methods of identification and describe several painless methods for marking in long-term experimental studies. The use of non-traumatic and non-invasive methods requires the renewal of marks as they fade and additional handling of the rodents. Laboratory personnel must be trained in stress-minimizing handling techniques to make mark renewal less stressful. The individual marking of animals and avoidance of misidentification are of great importance for ensuring the success of preclinical studies. Currently, appropriate anesthesia and the reduction in laboratory animal suffering during experiments are widely regulated. However, not enough attention is given to minimizing rodent discomfort and pain during marking, although a wide range of different methods of animal marking is available to researchers and vivarium staff ,2. AnimaIt is well known that all marking techniques are brief procedures restraining the animals, resulting in some degree of discomfort or pain. Since these procedures are performed on a huge number of rodents, even small improvements in the procedures can lead to a significant improvement in the overall effect. Ear punching and notching, toe clipping, and tattooing are frequently used for the permanent marking of laboratory rodents. At the same time, research on the well-being of animals showed that ear punching and tagging may cause pain ,4. FolloSince rodent marking is an essential part of laboratory research, additional attention should be given to non-invasive marking methods to ensure animal welfare. In this review, we aimed to describe a wide range of marking methods in order to raise awareness of possible ethical non-invasive options. Importantly, we also highlight ways to increase the reliability of non-invasive methods and the possibility of implementing non-invasive marking in long-term experiments with the use of stress-minimizing rodent handling.According to the degree of intervention to the bodies of laboratory rodents, we suggest classifying all marking methods into traumatic, invasive, and non-invasive.Traumatic methods are associated with the disruption of animal bodily integrity and include ear tagging, punching, and notching, as well as toe clipping.Some guidelines recognize that particular traumatic methods of individual identification should be used only when absolutely necessary or when no other forms of marking are possible or feasible ,9, with An important issue to be mentioned is that ear tagging may create an entry portal for pathogens and result in staphylococcus infection, and metal tags may cause auricular chondritis due to the release of iron and copper ions from the tag in mice ,11. CompToe clipping is one of the widely used marking methods that additionally allows for the collection of biomaterials for genotyping. Some studies reported neither significant corticosterone (pain and stress marker) elevation in 7-day-old mice after toe clipping , nor a hInvasive methods do not disrupt animal bodily integrity but still comprise procedures when the body is injected with an ink or implanted with a special device. Invasive methods include tail, toe, and ear tattooing, and the implantation of transponders for Radio Frequency Identification (RFID). We also include freeze marking in this group of methods since it affects the pigment-producing function of melanocytes in rodent skin.The tattooing may be performed by using special electronic tattooing machines or manually. Some reports showed that tail tattooing caused significant tail inflammation in mice, as well as agitation and anxiety , and theFreeze marking relies on the ability of low temperatures to destroy pigment-producing cells in the animals’ skin, resulting in fur discoloration. It provides permanent identification, but can only be used on dark-coated animals and can cause scarring in the case of excessively prolonged cold impact .Despite being one of the most advanced and sophisticated rodent marking systems that additionally allows for the tracking of animal behaviors , the impNon-invasive methods are of the highest ethical relevance since they require only painless manipulations, such as fur clipping, fur shaving, fur or skin dyeing, bleach marking, or biometry based on ear blood vessel patterns  14,22,2,222,23.However, there are concerns that non-invasive marking methods are less reliable, which may result in misidentification or require additional handling for repeated marking. We suppose that the use of a combination of several non-invasive marking methods  may enhance the robustness of non-invasive rodent marking . A combiOne of the principal questions is whether non-invasive methods may be modified to allow reliable individual identification in long-term experiments and replace invasive and traumatic methods. Even though non-toxic markers that do not disappear within 6–12 weeks are commercially available ,25, the Even ethically acceptable non-invasive methods of individual animal marking may have limitations. The odor of markers used for tail identification may be aversive to rats . In addiThe use of a system for the biometric identification of rodents based on patterns of ear vessels has also been reported . BiometrOne of the concerns arising in respect to ethical non-invasive marking methods is the potential need for more frequent handling and restraining, which may cause additional stress in animals. Indeed, it was shown that handling results in elevated levels of corticosterone in mice, suggesting increased stress . Even thThe general rule is to handle animals calmly yet firmly, so that they become accustomed to human contact without feeling threatened or stressed. The habituation of mice to the experimenter also has a positive effect on the reduction in stress levels from handling . TraininA substantial body of research is devoted to the investigation of the impacts of different handling techniques on anxiety- and stress-like behavior in laboratory rodents. Thus, it was shown that tunnel handling resulted in lower anxiety in comparison to tail handling ,32. SomeFinally, it is essential that handlers are properly trained in how to handle and restrain mice and rats with minimal stress to the animals. This includes learning how to recognize signs of distress in rodents  as well as understanding how different types of restraint devices should be used correctly without causing pain, harm, or discomfort . TraininApproaches to the minimization of laboratory animal suffering and the implementation of the 3Rs principles are being widely discussed ,35. ImprFrom an ethical point of view, standards of working with laboratory animals should emphasize the importance of non-invasive marking methods. In reality, this task is closely related to the development of new methods of reliable non-invasive marking that could ensure long-term identification. Biometric identification, as described above, represents a promising example of a non-invasive method of permanent identification . Thus, fNowadays, commonly used non-invasive marking methods are non-permanent and require the renewal of marks in long-term experiments, which means additional handling and restraint. Although this approach seems more ethical, the comprehensive training of staff in stress-minimizing handling and restraint techniques is required.It is important to highlight several factors that may influence the longevity of non-invasive, non-permanent marks. These factors may include environmental conditions, animal behaviors, and marking techniques. Environmental factors, such as sunlight exposure and abrasion, can accelerate the fading or removal of marks. Animal behaviors, such as grooming or rubbing against objects, can also affect mark durability. In addition, the choice in marking technique and materials used can have a significant impact on mark longevity.The length of the study period determines the number of times that marks need to be refreshed; therefore, longer studies may require more frequent refreshing to maintain identification accuracy. The visibility of the mark should be evaluated periodically to determine the need for refreshing. This can be achieved through visual inspections, remote sensing technologies, and cameras. Monitoring the rate of mark deterioration helps to estimate the optimal refresh frequency. The regular assessment of temporary mark legibility and durability allows researchers to adjust the refreshment schedule accordingly.We hypothesized possible factors hampering the implementation of non-invasive methods of rodents marking .Concerns about the lower reliability of non-invasive methods is one of the main reasons for the lack of wider use of non-invasive methods. Indeed, the dyes used for marking may fade or may be washed off during grooming. However, this fear can be overcome with constant monitoring of the animals, refreshing the dye as it fades, and the use of a combination of non-invasive methods.Concerns about inapplicability for long-term experiments: Marker and dye marks may fade over time, and shaved or bleached hair can grow back. However, the renewal of non-invasive marks using stress-minimizing handling and restraint techniques may allow non-invasive marking in long-term experiments.Stress related to additional handling during the renewal of non-invasive non-permanent marks: The problem of handling-related stress in rodents is widely recognized. At the same time, non-aversive methods of rodent handling, such as tunnel handling, may reduce stress. There is a need to conduct long-term studies dedicated to the question of welfare in rodents receiving traumatic/invasive permanent marks in comparison with rodents receiving non-invasive marks and more frequent handling.Insufficient financial resources are one of the fundamental causes that limit the implementation of best laboratory practices. The use of ethical non-invasive marking methods requires additional time for the refreshment of dyes or bleach marks, which implies additional working hours for laboratory staff that need to be paid. The development and implementation of novel methods based on biometry also requires financial support.Insufficient staff training: The problem of insufficient staff training is closely related to the lack of financial resources that would allow for the education and familiarization of specialists with ethical and novel approaches to animal marking. Moreover, hiring additional special staff dedicated solely to animal care and support with experimental procedures would reduce the amount of work for research staff and help to achieve higher standards of animal welfare.Perception of animals as just objects: In some cases, laboratory rodents are perceived only as tools to achieve a scientific goal without prioritizing their welfare . In suchThe use of non-invasive methods of rodent marking may be promoted by raising awareness of the feasibility of such methods if they are applied with due precautions or in a combined manner. Bioethical education among researchers may also aid in changing perceptions of laboratory animals and to raise empathy toward them. Legal regulations regarding acceptable methods of animal marking play an important role in the development of a culture of applying a particular method. Regulations governing animal research should contain clear rules regarding choosing particular types of marking with a focus on the most ethical approaches. A list of exemptions from the ‘most ethical marking rule’ should be clearly defined and explicitly establish cases where it is possible to use invasive or traumatic marking methods. Legal regulations regarding animal marking should be known to all laboratory staff, and compliance with such rules should be rigorously monitored.To summarize the above, in striving to achieve the highest ethical standards, researchers should minimize rodent discomfort, pain, and possible health complications even at the marking stage. However, this task requires a complex approach that includes changing researchers’ perception of animals, familiarity with the possibility of using non-invasive marking methods with due precautions, and the development of new reliable non-invasive marking techniques and associated marking reagents. The most difficult and long-term tasks include achieving higher financial support to provide training for laboratory staff and payments for the additional time required for the renewal of non-invasive marks, as well as the development of novel methods of rodent marking.The use of non-invasive marking methods may help to avoid health complications related to some methods of traumatic and invasive marking . Importantly, non-invasive methods allow the avoidance of pain at the marking stage. However, one of the serious limitations of non-invasive marking is represented by the need for additional handling for mark renewal, which bears the potential for additional stress. Novel studies are needed to compare long-term welfare parameters  in rodents marked in a traumatic/invasive manner and in rodents receiving non-invasive marks and stress-minimizing handling required for mark renewal.The reduction in rodent discomfort, pain, and health complications even at the marking stage is an important bioethical task that does not require less attention than the reduction in animal pain during experiments. The use of non-invasive painless methods of marking is limited due to fears of the unreliability of the method. In this paper, we described various non-invasive methods of rodent marking and ways of improving their reliability. We aimed to raise awareness of the possibility of painlessly marking rodents without the fear of mixing up individual animals. There is a need to spread knowledge on painless non-invasive methods of marking.There is a need for the development of new bioethical yet reliable methods of identification. At the moment, we suggest the use of a combination of already established non-invasive methods to avoid concerns about mixing up individual animals. The renewal of non-invasive temporary marks in long-term experiments also seems a more ethical option compared to traumatic and invasive marking; however, rules of stress-minimizing handling and restraint should be followed. Using a combination of non-invasive marking methods and renewal of marks as they fade requires more time from laboratory staff, but such efforts are justified, given that animals make a tremendous contribution to research and deserve the most humane treatment. Additional studies are required to compare welfare in rodents that receive traumatic/invasive marks and in rodents receiving non-invasive marks and additional handling for marks renewal.


Infertility has been recognized as a civilizational disease. One of the most common causes of infertility is polycystic ovary syndrome (PCOS). Closely interrelated immunometabolic mechanisms underlie the development of this complex syndrome and lead to infertility. The direct cause of infertility in PCOS is ovulation and implantation disorders caused by low-grade inflammation of ovarian tissue and endometrium which, in turn, result from immune and metabolic system disorders. The systemic immune response, in particular the inflammatory response, in conjunction with metabolic disorders, insulin resistance (IR), hyperadrenalism, insufficient secretion of progesterone, and oxidative stress lead not only to cardiovascular diseases, cancer, autoimmunity, and lipid metabolism disorders but also to infertility. Depending on the genetic and environmental conditions as well as certain cultural factors, some diseases may occur immediately, while others may become apparent years after an infertility diagnosis. Each of them alone can be a significant factor contributing to the development of PCOS and infertility. Further research will allow clinical management protocols to be established for PCOS patients experiencing infertility so that a targeted therapy approach can be applied to the factor underlying and driving the “vicious circle” alongside symptomatic treatment and ovulation stimulation. Hence, therapy of fertility for PCOS should be conducted by interdisciplinary teams of specialists as an in-depth understanding of the molecular relationships and clinical implications between the immunological and metabolic factors that trigger reproductive system disorders is necessary to restore the physiology and homeostasis of the body and, thus, fertility, among PCOS patients. Infertility is a growing social problem, which is why the World Health Organization (WHO) recognized it as a civilization disease of the 21st century. Although infertility is defined as the inability to conceive despite 12 months of regular sexual intercourse with the intention of having a child in women <35 years of age (American Society for Reproductive Medicine—ASRM), often a term of 6 months of unsuccessful attempts in women >35 years of age is applied in clinical practice within the similar diagnostic management .Infertility is not a uniform entity resulting from a dysfunction exclusive to the reproductive system but a multifactorial one. More and more attention is being paid to the action of the systemic enzymatic response that is dependent on the inflammatory response in connection with functional disorders, insulin resistance (IR), and oxidative stress. They are not only present in cardiovascular diseases but also in infertility . In termPolycystic ovary syndrome is a common disease with over-representation in infertile women. PCOS contributes to up to 56% of infertility cases ,8. The dThe results of the analysis of PCOS prevalence in 204 countries in the years 1990–2019 were published in 2022. In 2019, the global point and annual age-standardized PCOS incidence rates increased by almost 30% since 1990 . AccordiThe etiology of PCOS is multifactorial. The disorders occurring in this endocrinopathy are the result of a complex interaction of genetic, epigenetic, gender, racial, environmental, and stochastic factors. PCOS not only affects the reproductive system but the entire body and accompanies the patient throughout their life . In the One of the links that have been recognized in the pathogenesis of PCOS is insulin resistance (IR), which is induced by inflammation and hormonal dysfunctions originating in adipose tissue. Abnormal glucose and lipid metabolism cause low-grade inflammation in the endothelium. On the other hand, the presence of chronic inflammation may contribute to the development of IR, which is enhanced by adipokines released from adipose tissue . IR alsoIt has long been known that there are genetic factors associated with the inheritance of PCOS . HoweverIt should be emphasized that the pathomechanism of infertility in PCOS begins to be regulated on the gene level . StudiesIt has been proven that relatives of women with PCOS, both male and female, are also at increased risk of developing not only reproductive but also metabolic disorders associated with PCOS, such as a higher incidence of insulin resistance, obesity, diabetes, cardiovascular disease, early male pattern baldness, and hypertrichosis .This seems to be an obvious consequence since the abnormality underlying PCOS is indeed an aberration in the gonadotropin/steroidogenesis/insulin pathway associated with inflammation. Thus, ovarian dysfunction in the form of PCOS can only be a consequence of this and one of many further symptoms. Among the studies confirming the influence of hereditary factors on the occurrence of PCOS that merits attention is a Swedish study conducted on a group of over 29,000 women. It was shown that the daughters of women with PCOS had a five-fold increased risk of developing this syndrome than the daughters born to women without PCOS . In a DuAlthough the role of hereditary factors in the occurrence of PCOS and infertility is widely recognized, intensive research is still being conducted on the genetic aspect of infertility in this endocrinopathy. Genome-wide association studies (GWAS) have increasingly identified a number of regions of the human genome that may be responsible for the genetic background of PCOS .In a study on the link between ovarian cancer and PCOS, Zou et al., identified key genes associated with PCOS and ovarian cancer. Using integrated bioinformatics analysis, they found as many as 1061 differentially expressed genes (DEGs) in PCOS patients compared to normal women based on the GSE34526 dataset from the GEO database (GEO Expression Omnibus). In addition, they found 128 common genes with differential expression (DEG) in the progression of PCOS and ovarian cancer .One of the newer large-scale genome-wide association studies (GWAS) linking more than 10,000,000 genetic markers in 10,074 European women with PCOS and 103,164 controls revealed three new loci  associated with PCOS risk. The data also provided the first genetic evidence for a male PCOS phenotype associated with male pattern baldness and a causal relationship with depression, which had previously only been reported on the basis of observational studies of these patients. This international meta-analysis of GWAS studies has provided evidence of a common genetic basis for many different symptoms in PCOS, including gender, mental health, and reproductive potential .Steroidogenesis pathways are an important aspect in the etiology of infertility as they are associated with genetic variants that cause clinical hypothalamic-pituitary and gonadotropin receptor dysfunction in PCOS women with infertility problems.The genes involved in the steroidogenesis process include those from the CYP family: CYP11A, CYP21, CYP17, CYP19, the AR-gene of the androgen receptor as well as the gene encoding sex hormone binding globulin (SHBG) .The disruption of the transcription of these genes leads to hyperandrogenism and ovulation disorders. Among the CYP genes (encoding polypeptides of the cytochrome P450 family), the CYP 19A1 gene is responsible for the activity of aromatase p450, which is also necessary for the formation of estrogens. A defect in this gene causes a decrease in the activity of this enzyme, which leads to hyperandrogenism in both obese and lean women with PCOS. The result of this finding suggests that CYP19A1 contributes to infertility through the adverse effects of androgens on metabolism and inflammation impairing ovulation.Other genes from the CYP subset are also extremely important in the context of the development of PCOS and infertility. CYP17A1 encodes an enzyme that catalyzes the production of steroidogenesis: androgens, glucocorticoids, and progestins. Mutations in this gene are associated with adrenal hyperplasia, pseudohermaphroditism, and deficiencies of enzymes responsible for changes in the androgen pathway, including progesterone deficiency. In contrast, the CYP11A1 gene encodes a protein found in the inner membrane of mitochondria that catalyzes the conversion of cholesterol to pregnenolone in the first step of steroidogenesis .The previously described metabolic disorders in PCOS that are associated with infertility, such as IR, have also been linked to polymorphisms in specific genes. CAPN10, which encodes calpain 10 and is involved in insulin function and secretion, has been identified as a risk gene for type 2 diabetes, and specific CAPN10 variants have been associated with PCOS . SimilarAmong the genes that have been discovered and associated with fertility disorders in PCOS, the following should also be mentioned: the follicle-stimulating hormone polypeptide B gene (FSHB), the luteinizing-hormone gene/gonadotropin receptor (LHCGR), the follicle-stimulating hormone receptor gene (FSHR) encoding G-protein coupled receptors essential for the development of the gonads, the androgen receptor gene (AR), the anti-Müllerian hormone (AMH) gene, the DENND1A gene encoding a protein called connecdenn-1 that transports the endosomes contained in the theca cells of the ovary, the overexpression of which is the source of increased androgenization. In addition, SRD5A, a variant of the 5α-reductase type 1 gene responsible for hirsutism, as well as other genes related to metabolism, such as thyroid adenoma-associated (THADA) gene or the already mentioned INSR-insulin receptor gene, associated with insulin resistance PCOS in liver cells, fibroblasts, and skeletal muscle, including also insulin receptor substrate protein (IRS) ,38.Among them, the AMH gene deserves special attention. It encodes a protein that is involved in infertility. Whole exome sequencing and GWAS identified rare variants of the AMH gene that have been linked to PCOS. Reduced AMH signaling has been shown to result in a lack of inhibition of CYP17 activity, resulting in increased androgen biosynthesis and impaired fertility . TherefoSubsequent studies have shown that polymorphism in the FSHR gene is significantly associated with PCOS. Two missense mutations were detected, including p.Ala307Thr and p.Asn680Ser. These polymorphisms of the FSHR gene have been shown to have a significant impact on the hormonal response in the ovaries. FSHR mutation rates were also shown to be significantly higher in women with PCOS than in controls .In addition, it has been demonstrated that infertility in women with PCOS may be associated with the occurrence of changes in the HOXA-10 and HOXA-11 genes, which leads to implantation failure as endometrial receptivity is disturbed. HOXA10 promotes cell proliferation during decidualization , during Additionally, of great importance was the discovery that bone morphogenetic protein (BMP), a growth factor in the transforming growth factor-beta (TGF-beta) superfamily, plays an essential role in regulating granulosa cell proliferation and oocyte development. In addition, BMP plays an important role in the process of embryo implantation in the uterine cavity. The loss of BMP signaling in this tissue is due to the BMPRIB mutation and leads to abnormal endometrial gland formation. BMP is essential for estradiol biosynthesis and cumulus cell expansion in vivo. The gene mutation for this protein is associated with a reduced level of aromatase production in granulosa cells, which is why this mutation seems to be one of the key factors responsible for infertility among women with PCOS ,44,46.Two polymorphisms of the plasminogen activator inhibitor-1 (PAI-1) gene are also very important in the context of the development of PCOS and infertility: −675 5 G > 4 G (rs1799889) and −844 G > A (rs2227631), which are associated with increased transcriptional activity and PAI-1 protein levels . PAI-1 iAdditionally, although almost half of the people have the PAI-1 polymorphism in the context of obstetric failures and the coexistence of other unfavorable factors acting synergistically, it is worth evaluating this gene among women with PCOS . Even inComplete PAI-1 deficiency is caused by mutations in the SERPINE1 gene, which is located on chromosome 7 (7q21.3-22) and consists of nine exons . PlasminIn the presence of specific polymorphisms of the PAI-1 gene, this protein is overproduced, as a result of which the ovarian plasminogen-plasmin pathway is disturbed. Plasminogen is not converted to plasmin upon follicle rupture and, therefore, does not lead to ovulation in women with PCOS . In addiOne large meta-analysis spanning almost 33 years investigating the role of the PAI-1 gene in adverse pregnancy and gynecological complications described the complex role of the PAI-1 gene in various reproductive failures, including recurrent pregnancy loss (RPL), pre-eclampsia (PE), gestational diabetes (GDM), impaired fetal growth (FGR), repeated implantation failure (RIF), polycystic ovary syndrome (PCOS), and endometriosis . The PAISome studies have shown that PAI-1-844G/A polymorphism is associated with an increased risk of developing metabolic syndrome . TherefoInterestingly, the detection of the presence of the PAI-1 polymorphism carries with it important clinical decisions regarding therapy, because overexpression of this protein can be treated, among others, through the use of metformin .PCOS phenotypes are highly heterogeneous, but more and more reports are explaining their origin. PCOS is associated with changes in many molecular factors, not only gene defects but also multiple polymorphisms or nucleotide changes that may interfere with the transcriptional activity of genes .From the studies presented so far, it is clear that some of the genes are overexpressed and manifest clinically, while others are not, depending on the exposure of the body to environmental factors from the moment of conception . GrowingOn the one hand, it has been proven that the same genes that are involved in the pathomechanism of oxidative stress, lipid metabolism, or insulin signaling are involved in follicle growth arrest and metabolic disorders associated with various PCOS phenotypes. On the other hand, it has been shown that these diverse clinical presentations of PCOS often result not from changes in the DNA sequence itself, but from transgenerational and mitotic hereditary processes, that is, epigenetic changes. Tissue-level epigenetic changes are responsible for changing the phenotype of cells as a response to a changed environment. Chromatin modification, even without additions or deletions to pre-existing DNA, is the trigger for epigenetic reprogramming. Specific mechanisms are involved in this process, such as methylation, hydromethylation, formylation, or carboxylation. DNA methylation is an enzymatic reaction that consists of adding a methyl group, usually at the five carbon of the cytosine pyrimidine ring, and then guanine, which are called dinucleotides. In addition to PCOS, the clinical significance of DNA methylation has been demonstrated in such diseases as cancer, type 2 diabetes, neurodegenerative diseases, and cardiovascular diseases .Women with PCOS have changes in DNA methylation in peripheral and umbilical blood, suggesting a link between the PCOS phenotype and epigenetic changes in systemic and fetal circulation cells . ChangesOther studies show the observation of various clinical and functional symptoms in PCOS and their occurrence at different ages, depending on the therapeutic interventions, which was associated with phenotypic changes in the affected tissues ,60.Knowledge about the possibility of using genetic diagnostics in PCOS may prove very useful in the treatment of infertility in these patients. The ability to influence the modification of DNA methylation, gene expression, and the appropriate phenotype opens up new ways for clinicians to select therapy.Ovulation disorders are a key mechanism of infertility in PCOS. Restoration of this process is the basic element of infertility treatment among these women, but it turns out that the genetic background is important for the success of this therapy. Treatment with metformin is recommended as a first-line drug to induce ovulation in PCOS patients. Metformin has been shown to regulate DNA methylation by controlling the activity of S-adenosylhomocysteine hydrolase, an S-adenosylhomocysteine hydrolyzing enzyme that inhibits DNMT activity . HoweverIt should be emphasized, however, that genetic tests are not widely available and their costs often limit the possibility of using them in all women with PCOS and infertility. In addition, to a large extent, the clinical picture and the phenotype of a specific PCOS patient will depend not only on the genetic background but also on the genome modifications resulting from the epigenetic changes occurring throughout her life, starting from the moment of conception .Many women with PCOS are overweight or obese, depending on the different phenotypes of the syndrome . The preObesity, especially defined by waist circumference, has been confirmed to have a significant effect on fertility, triggering ovulatory disorders and resulting in androgen excess . ObesityAn extensive meta-analysis of 30 studies has shown that excess body weight is associated with more severe PCOS symptoms and significantly worsens all metabolic and reproductive outcomes .Adipose tissue can be considered as an endocrine gland that secretes bioactive peptides (adipokines) that act locally, paracrinely, autocrinely, and systemically through the production of hormones, which may play a key role in infertility with PCOS. Research on adipokines has revealed an important link between the basic factors underlying the pathomechanism of PCOS, such as abdominal obesity, IR, hyperandrogenemia, inflammation, oxidative stress, and reproductive disorders ,77. It sAs adipose tissue increases in obesity, the pro- and anti-inflammatory properties of adipokines that are found to be in balance in lean and healthy individuals shift towards pro-inflammatory mediators . InteresIn one of the latest large-scale reviews of original papers listed in the PubMed database, the activities of individual adipokines were reported to affect the development of PCOS, among others .Another cytokine that is related to PCOS pathogenesis is chemerin. A meta-analysis of a total of 897 participants  confirmed that circulating chemerin concentrations were higher in PCOS patients than in the control group . ChemeriIn PCOS patients, IR has been shown to increase leptin secretion from white adipose tissue . On the Among PCOS patients, the expression of the aromatase gene in granulosa cells is reduced by mitogen-activated protein kinase (MAPK) and PI3K, which is an expression of leptin resistance. Therefore, in patients with PCOS, the concentration of leptin in follicular fluid increases and may cause ovulatory disorders . Kucera Follistatin and visfatin are two adipokines whose effects on the reproductive system are twofold. Follistatin has anti-angiogenesis properties. It helps to prevent the onset of metabolic diseases by inhibiting the immune system factors from the transforming growth factor (TGF) superfamily. Follistatin contributes to the growth of brown adipose tissue, which prevents obesity and its pro-inflammatory effects in the body .Many studies clearly show that visfatin is elevated in the adipose tissue of obese patients. One meta-analysis of 13 observational studies of visfatin and overweight/obese patients involving 644 subjects showed that plasma visfatin concentrations were higher in persons who were overweight/obese, with DM2, metabolic syndrome, and cardiovascular disease . NumerouThe APM1 gene, which encodes adiponectin, is located on chromosome 3q27, close to the site responsible for DM2 and obesity . Hence, Adiponectin receptors are also found in the central nervous system, as well as on many reproductive system organs: the ovaries, fallopian tube, endometrium, and testes. Human granulosa cells in ovarian follicles have receptors for this protein, which regulate the process of angiogenesis in the ovaries and the synthesis of sex hormones. It has also been demonstrated that adiponectin affects the release of gonadotropin, the normal course of pregnancy, as well as the outcomes of assisted reproduction technologies .Hypoadiponectinemia, through its action reducing IR, atherogenic damage, and inflammation of the vascular endothelium, contributes to disorders leading to PCOS . One stuThe presence of central obesity has been shown to be a risk factor for anovulatory cycles in women with PCOS compared to ovulatory PCOS and corresponding controls despite a similar BMI . The stuIt has been shown that adiponectin inhibits the secretion of LH and androgens, thereby promoting ovulation. With its reduced concentrations, there is an increased secretion of LH in the pituitary gland and overproduction of androgens in the theca cells of the ovary, which leads to ovulation disorders. Adiponectin, by activating the PI3K-AKT pathway, reduces IR, hyperandrogenemia, which not only regulates the metabolic system, but also promotes the proper functioning of the ovaries ,114.Omentin is produced primarily in visceral adipose tissue and is credited with insulin-sensitizing and anti-inflammatory effects. Tang et al., reviewed 13 studies conducted among women with PCOS and showed that circulating levels of omentin-1 are significantly lower in women with PCOS compared to the controls. The findings showed that women with PCOS had elevated levels of interleukin 6 (IL-6), interleukin 18 (IL-18), TNF, retinol-binding protein 4 (RBP-4), resistin, leptin, insulin, LH, testosterone, free testosterone, and Homeostatic Model Assessment (HOMA), but they also had lower omentin-1, ghrelin, and Quantitative Insulin Sensitivity Check Index (QUICKI) values compared to healthy controls ,116. OrlOmentin regulation at the ovarian level differs from systemic regulation. In a cohort of women affected by infertility, the expression of chemerin and omentin in follicular fluid and granulosa cells (GC) was selectively increased in both the lean and obese PCOS groups . SimilarInsulin resistance, oxidative stress, and low-grade inflammation and their impact on ovulation disordersInsulin resistance is one of the main features of PCOS, characterized by a reduced cellular response to the physiological concentrations of insulin, which triggers a state of hyperinsulinemia . The mecWomen with the conventional PCOS phenotype have been shown to be obese and have higher insulin and IR concentrations . It shouAlthough PCOS has been proven to be associated with IR independently of obesity, excess body weight significantly increases both its incidence and severity . In manyInsulin resistance causes hyperglycemia and increased levels of free fatty acids (FFAs). In turn, FFAs, by hyperactivating the electron transport chain, stimulate mononuclear cells to release reactive oxygen species (ROS), which in turn induce oxidative stress . FurtherIt has also been shown that the presence of antioxidants in follicular fluid directly correlates with the success of in vitro fertilization, and superoxide dismutase is involved in the process of embryo implantation in the endometrium. Oxidative stress in the oocyte negatively affects both its meiotic and cytoplasmic maturation, generating oocytes of reduced quality, and inducing abnormalities in the development of the embryo before implantation .Suppression of oxidative stress leads to a reduction in apoptosis and has therapeutic potential in this regard, most likely by protecting mitochondrial function and regulating the proteins associated with programmed cell death . Mitocho2) consumption, glutathione, MMP, and mitochondrial membrane potential, impaired oxidative phosphorylation complex (OXPHOS) activity, as well as a significant increase in the production of ROS [Mitochondrial dysfunction in the leukocytes of women with PCOS and IR is manifest in a decrease in mitochondrial oxygen (On of ROS . Besidesn of ROS .2O2) activity. Therefore, in PCOS, increased LH stimulation leads to an increase in the amount of ROS, thus, generating excessive oxidative stress [Other studies have shown that the presence of oxidative stress associated with increased production and accumulation of ROS in the female reproductive system negatively affects folliculogenesis and oogenesis. On the one hand, the very presence of ROS is essential for proper folliculogenesis because some of their forms are involved in signaling cascades, such as nuclear factor kappa B (NF-kB) and vascular endothelial growth factor (VEGF), which are necessary for follicle growth. On the other hand, excess ROS can lead to impaired oocyte quality due to mitochondrial dysfunction, decreased ATP production, oocyte aging, and oocyte/embryo aneuploidy . Additioe stress . Turan ee stress .In a study of Iranian women with PCOS, it was demonstrated that they had increased oxidative stress, as measured by the Advanced Protein Oxidation Products (AOPP), and reduced antioxidant capacity compared to healthy controls matched for age and BMI. AOPPs were recognized as new markers of oxidant-mediated protein damage and function as a new class of pro-inflammatory mediators. Much higher levels of AOPP and a much lower total antioxidant status may contribute to the increased risk of cardiovascular disease in women with PCOS, in addition to the already known risk factors, such as IR, hypertension, central obesity, and dyslipidemia .One study also showed that the pathophysiology of PCOS is a significant contributor to oxidative stress, independent of IR and BMI. In follicular fluid and granulosa cells, 71 women with PCOS and 50 control women undergoing in vitro fertilization (IVF) were tested for the activity and levels of redox markers and transcript expression, respectively. Superoxide dismutase, glutathione reductase, glutathione peroxidase, and paraoxonase-1 (PON1) activity were significantly lower in the follicular fluid of women with PCOS than in the control group . Özer et2O2)-induced cytotoxicity and cell apoptosis by inhibiting the expression of the pro-apoptotic Bax protein [The oxidoreductive stress occurring in PCOS should be viewed in many dimensions. It turns out that adipokines also play a significant role here. The protective effect of omentin against oxidative stress was determined by quantifying the reactivity of antioxidant enzymes. In studies by Shi et al., it was shown that omentin can attenuate hydrogen peroxide  concentrations . IncreasAndrogen excess, one of the key characteristics of women with PCOS, also inhibits glyoxylase I activity . ReducinNumerous reports have also indicated the existence of an opposite mechanism wherein it is possible that AGEs play a key role in enhancing androgen biosynthesis in PCOS patients. This is mediated not only by the previously described direct enzymatic change but also by IR and inflammatory induction. Serum AGE levels in women with PCOS have been shown to be positively correlated with testosterone levels, regardless of BMI . MouanneAnalyzing the individual elements of the pathomechanism leading to the formation of PCOS, it can be seen that they ultimately lead to an imbalance in the immune system. In PCOS, the key fact is that ovulation is a condition dependent on the occurrence of local, self-limiting inflammation . The infThis normal, two-stage course of ovulation is disrupted by the pathological, chronic inflammation of the ovary caused by IR and hyperandrogenism . It direChronic systemic inflammation has been confirmed to exist among women with PCOS . C-reactIt turns out that the success of reproduction involving follicle growth, oocyte maturation, and successful ovulation and implantation- is based on processes that are ultimately regulated at the immune system level . CytokinIt has been shown that almost 30% of women with PCOS experiencing ovulatory disorders are resistant to stimulation with clomiphene citrate, which induces follicular growth through the action of FSH. Among these patients, there was a positive correlation between the serum levels of the pro-inflammatory chemokine CXCL16 and the inflammatory protein CRP. CXCL-16 may impair the ovarian response to clomiphene citrate by increasing angiogenesis and inflammation , thus, bThe systemic inflammation in PCOS caused by IR and lipotoxicity, which are often associated with obesity or visceral adipose tissue dysfunction, can be transmitted to the ovary and follicular fluid by triggering cytokine imbalances. Optimal cytokine expression is generally required for normal ovarian function. However, increased or abnormal secretion of pro-inflammatory cytokines at inappropriate time points during the cycle impairs the normal course of ovulation and fertility. This is because ovulation is an inflammatory process during which a large pool of leukocytes is recruited to the preovulatory follicle wall .In contrast, systemic, chronic inflammation triggered by PCOS may interfere with the regulation of this locally ongoing inflammatory process during ovulation. In response to the LH signaling from the periovulatory surge by the pituitary gland, granulosa cells begin to secrete steroid hormones and inflammatory mediators necessary to initiate the rupture of the ovulatory follicle wall. They include prostaglandins, chemokines, and cytokines, which also have a chemotactic effect on leukocytes and other cells of the immune system that begin to flow into the ovarian tissue . In the Therefore, understanding these subtle mechanisms may contribute to improving the treatment of infertility due to ovulatory disorders in women with PCOS resulting from abnormal leukocyte function. The administration in stimulated cycles of not only the so far widely used HCG, which mimics the pituitary LH surge, but also of GM-CSF, may enhance the recruitment of leukocytes to the follicle wall and the normal wall rupture process. Leukocytes recruited to the periovulatory follicle in response to chemokines secreted by luteinized granulosa cells induce the angiogenesis and luteinization of subsequent granulosa cells, promoting the process of ovulation . SimilarQin et al., showed that the ovarian leukocytes of women with PCOS have a different cytokine profile compared to healthy controls . Using fAn extremely important aspect of the infertility etiology of women with PCOS is the low level of progesterone generated by the absence of or rarely occurring ovulation. Progesterone not only ensures the cyclic exfoliation of the endometrium and the occurrence of menstruation but, above all, is an immunomodulatory factor . As it tPCOS is characterized by the central dysregulation of the HPO axis with a rapid pulsation of GnRH, which is followed by a rapid pulsation of LH. This, in turn, impairs FSH-dependent follicular growth and generates anovulatory cycles with a chronic progesterone deficiency. Subsequently, there is the effect of an overproduction of androgens by the theca cells of the ovary and the failure of FSH-dependent aromatase. There is an increased release of androgens from the ovary into the bloodstream and, consequently, IR and inflammation, as well as clinical symptoms specific to PCOS, such as hirsutism, acne, androgenetic alopecia, and ovulation disorders .In a properly functioning ovary, the periovulatory release of the LH from the pituitary induces a transient increase in the expression of the progesterone receptor in the granulosa cells of the preovulatory follicles. Following the LH action, a portion of the granulosa cells of the ovulating follicle is gradually luteinized, releasing pre-ovulatory progesterone, which has a paracrine effect. It has been shown that progesterone is an important mediator of ovulation as it affects the regulation of genes located in the granulosa cells of ovulatory follicles, for example, those encoding proteases a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMST1) and cathepsin L (CTSL). Proteolytic enzymes digest the proteins of the follicle wall and help in its disintegration during ovulation . It has The exact mechanisms by which progesterone regulates ovulation are very complex. Its flagship effect is the effect on the release of cytokines and the regulation of immune system genes that affect the recruitment of neutrophils immediately before ovulation. The influx of neutrophils into the follicle wall is necessary for the further stages of rupture of the ovulatory follicle wall. Another important aspect is the proper activity of prostaglandin 2 (PGE2), which acts as an inflammatory mediator within the follicle, inducing the breakdown of the basement membrane, luteinization of the follicle wall, follicle rupture, and the release of the egg cell from the ovary. The enzyme that controls prostaglandin production is prostaglandin-endoperoxide synthase 2 (PTGS2), which is strongly induced by progesterone in ovarian granulosa cells in response to the LH surge .In addition, in PCOS patients, rare or abnormal ovulation implies an impaired receptivity of the endometrium and reproductive failures in terms of embryo implantation and placental development. It has been repeatedly shown that the immunomodulatory effect of progesterone in the endometrium promotes embryo implantation and pregnancy maintenance ,177. ProOvulatory disorders and tonic high estrogen levels in the presence of a progesterone deficiency result not only in endometrial infertility but also endometrial hyperplasia due to chronically elevated estrogen levels that are not counteracted by progesterone . ProgestThe immunomodulatory effect of progesterone is mediated by the progesterone-inducible blocking factor (PIBF) protein, which is produced in the presence of progesterone by progesterone receptor-positive lymphocytes. PIBF expression in lymphocytes shows an inverse correlation with NK cell activity. PIBF has been shown to increase the production of Th2 cytokines and inhibit NK cell degranulation . In womeExtremely important in reproductive medicine is the fact that progesterone, by binding to the glucocorticoid receptor (GR) in T lymphocytes, inhibits the production of pro-inflammatory IL-6 through dendritic cell-dependent pathways . TherefoProgesterone deficiency seems to be a key factor in the lack of control of autoimmune reactions triggered by estrogen and environmental factors, including specific autoantigens, including viral and bacterial infections. Progesterone, as a pro-pregnancy hormone, is necessary to sustain pregnancy which, in this context, is a semi-allograft. It triggers and maintains a specific, fetal-friendly environment in the endometrial cavity and in the mother’s system. On the one hand, it suppresses the rejection reactions of the fetus, whose antigens are half paternal and, therefore, foreign to the mother and, on the other hand, it triggers defense factors against infections to protect the newly developing body .Recently, attention has also been paid to the important role of progesterone in immunomodulatory reactions and in inflammation of the fallopian tubes. Progesterone regulates the morphology and function of the epithelial cells in the fallopian tube, enabling a normal pregnancy. It reduces the contraction of the fallopian tube muscles and the frequency of the ciliary rhythm, which affects the transport of the oocyte and embryo. In addition, an increase in its level in the first stage of ovulation enables the correct capacitation of spermatozoa .The multifaceted influence of various factors on the regulation of progesterone production, which contributes to the pathophysiology of such a complex syndrome as PCOS, is invariably important. It has been shown that the aforementioned adiponectin, the level of which is clearly reduced in PCOS patients, increases the secretion of progesterone and estradiol (E2), induced by IGF-1 in granulosa cells, by activating aromatase p450. Human ovarian granulosa cells have receptors for adiponectin. Thus, hypoadiponectinemia not only contributes to PCOS-related disorders by affecting IR and inflammation in the body, but it also reduces ovarian progesterone production ,185.Sexual dimorphism determines the directions of expression of cellular markers involved in innate and adaptive immunity. Women have better protection against infections, a predisposition to produce antibodies, and a predominance of the Th2 type response . The higProgesterone is a hormone that regulates key reproductive processes, from ovulation through implantation, placental development, and pregnancy. In addition, it has strong immunomodulatory properties that go beyond the reproductive system . In variIn addition, an association has been shown between PCOS and autoimmune diseases such as systemic lupus erythematosus (SLE) and Hashimoto’s thyroiditis ,193.Antinuclear antibodies (ANA) in systemic diseases have a direct negative effect on fertility at several stages of the reproductive process. The presence of ANA in a woman’s system significantly interferes with the development of the oocyte and embryo and, thus, impairs the process of fertilization, implantation, and the course of pregnancy. In addition, the precipitation of immune complexes at the maternal-fetal interface and complement activation with inflammatory infiltration may also occur. All of these mechanisms can lead to miscarriage or premature birth . StudiesIt turns out that in PCOS, the presence of many other autoantibodies has been documented in addition to ANA, antihistone, and anti-dsDNA,. Among them are: antisperm antibodies, anti-Sm antibodies , anti-carbonic anhydrase antibodies, anti-ovarian antibodies, and anti-pancreatic islet cell antibodies .This association of reproductive failure with a propensity for various non-organ-specific autoantibodies has been shown not only in women with PCOS ,197. EleThe relationship between antibodies against pancreatic islet cells and the development of PCOS cannot be overlooked either. In one study, these antibodies were found in over 80% of PCOS patients . The desOther studies among women with PCOS showed a 3-fold higher incidence of autoimmune thyroiditis (AIT). All three types of anti-thyroid antibodies: anti-thyroid peroxidase antibodies (anti-TPO), anti-thyroglobulin antibodies (anti-TG), and anti-thyrotropic receptor antibodies (TRAbs)—are associated with reproductive failure . The incThe association of autoimmunity with PCOS is undoubtedly an important factor in the development of the syndrome itself as well as the cause of infertility among women with this syndrome. More and more areas are sought where the diagnosis of PCOS should increase vigilance as to the possibility of developing comorbidities. One of the recent reports describes the coexistence of non-infectious uveitis with the occurrence of pro-inflammatory factors in PCOS in women of reproductive age .On the other hand, it is important to remember that autoimmunity itself can be a causal factor in PCOS. In autoimmunity, auto-reactive cells such as B and T lymphocytes are induced and antibodies are produced, which leads to the breakdown of the mechanisms responsible for self-tolerance . AntibodInfertility is associated with the suffering of individuals and affects the entire socio-demographic structure of the whole population. It results from immunological and metabolic abnormalities that initiate endocrine disorders. The main factors causing infertility among women with PCOS are impaired ovulation, implantation, and low-grade inflammation. On the one hand, IR and hyperandrogenism and, on the other hand, chronic inflammation, autoimmunity, and progesterone deficiency with a genetic and epigenetic background are the basis of fertility disorders and accompanying diseases in women with PCOS. Closely related immune-metabolic mechanisms underlie the development of this complex syndrome and cause infertility . PreviouRegardless of the presence of obesity and its obvious impact on the IR, a number of scientific studies suggest that women with PCOS exhibit an intrinsic form of IR that is unique to the disorder and contributes to infertility at the level of both ovulatory and implantation disorders.IR contributes to the development of the immunological processes that trigger inflammation, which has a negative impact on the processes taking place within the ovary. IR induces the overproduction of androgens in the ovaries through IGF-1 receptors, leading to hyperandrogenism and its aftermath.The influence of obesity on fertility may be associated with an increased leptin concentration, hypoadiponectinemia, a high concentration of triglycerides and free fatty acids in ovarian follicular fluid, and also the presence of oxidative stress and inflammatory mediators. This leads to abnormal ovulation and infertility.In recent reports, chronic, low-grade inflammation—regardless of the starting point—appears to be the most crucial factor contributing to the pathogenesis of PCOS and infertility.Oxidative stress present in the ovarian cells of PCOS patients has a negative impact on fertility, regardless of how the abnormal redox balance is triggered. It also points to a direct relationship between IR and immune system dysfunction by triggering oxidative stress and inflammation in the endothelium and, thus, also in the ovarian tissue.The effect of progesterone on the immune processes associated with ovulation, implantation, and pregnancy development confirms the key role of this immunomodulating hormone in reproduction. A chronic progesterone deficiency in women with PCOS, as well as hormonal and immunological imbalances, are unquestionably two of the most important causes of infertility in this syndrome.The balance between the effects of estrogen and progesterone in a woman’s body not only guarantees the proper hormonal balance that is conducive to maintaining fertility but also allows the inhibition of autoimmune reactions, also after supplementation in PCOS.The treatment of infertility in PCOS should be carried out by interdisciplinary teams of specialists so that patients are correctly diagnosed and the complex therapy successfully restores health and fertility.


Mitochondrial selective autophagy, known as mitophagy, is the primary cellular process through which cells regulate the quantity and the quality of mitochondria in response to metabolic and physiological changes. Mitophagy has been the subject of extensive research in the last two decades, uncovering the intricate signaling pathways and the complex mechanisms involved in degradation of superfluous and/or damaged mitochondria. Several mitophagy-related proteins have been identified, revealing a sophisticated regulatory network that preserves mitochondrial function upon developmental, hormonal, and environmental cues. Furthermore, the interconnected communication between these signaling pathways modulate energy metabolism promoting tissue and organ homeostasis. When mitophagy malfunctions, defective mitochondria are accumulated resulting in bioenergetic imbalance, elevated generation of reactive oxygen species (ROS), and chronic inflammation, ultimately leading to cellular death and tissue degeneration. Consequently, mitophagy significantly contributes to cellular physiology, tissue integrity, as well as overall organismal development, healthspan, and survival.Mitophagy in health and disease, Volume II in Frontiers in Cell and Developmental Biology includes a series of 4 articles that shed light on the diverse facets of mitophagy research. The following articles discuss recent advances in the field of mitophagy and mitochondrial metabolism research and provide novel insights into the regulatory mechanisms in various physiological and pathological conditions.The Research Topic on Akbari et al. provide an overview of the molecular mechanisms that orchestrate mtDNA repair and transcription underscoring the dynamic functional interactions between the components of these processes. The authors suggest that mtDNA-targeted modulation can be utilized for the development of novel therapeutic interventions to mitigate the negative effects associated with mtDNA instability.Despite its relatively small size, mitochondrial DNA (mtDNA) is crucial for normal organismal development, function, and survival. Loss of mtDNA integrity has been implicated in the normal aging process, inflammation, and the etiology and pathogenesis of several diseases, including mitochondrial diseases and premature ageing. In their review, Zheng et al. point out that excessive mitophagy might be detrimental for the myocardium in DCM. The authors suggest that the delicate balance between mitochondrial biogenesis and mitophagy is pivotal for the maintenance of cellular metabolism and cardiac function in individuals with diabetes. In addition, they highlight the need for a better understanding of the precise molecular mechanisms involved in mitophagy and the development of effective interventions to coordinate mitochondrial biogenesis and degradation, thereby preserving energy metabolism and ameliorating cardiac dysfunction in diabetic patients.Diabetic cardiomyopathy (DCM) is a significant cause of mortality in individuals with diabetes mellitus (DM) and is characterized by impaired heart function. While mitophagy is crucial for the elimination of damaged mitochondria and preserving cellular homeostasis, in their review, Zhang et al. emphasize the significance of mitophagy in mitigating the risk of DKD, especially in the context of additional pathological conditions, such as COVID-19 infection, which has been associated with an increased incidence of diabetic complications. Moreover, they underscore the crucial role of mitophagy in the maintenance of homeostasis in the glomeruli and tubules. They provide compelling evidence supporting the notion that mitophagy promotes energy metabolism and overall cellular function in the kidneys. The authors suggest that kidney function can be restored in individuals with DKD by enhancing mitochondrial degradation. Overall, the authors highlight the importance of mitophagy in kidney physiology and suggest its stimulation as a potential therapeutic strategy against DKD.Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease globally and a major microvascular complication associated with diabetes mellitus (DM). In their review, Schiavi et al. investigate the effects of oxygen and iron depletion on cellular and organismal physiology in both the nematode Caenorhabditis elegans and mammalian cells. Mild hypoxia and non-toxic levels of iron chelation promote cellular metabolism and enhance organismal survival by inducing mitophagy. The authors investigate the mitophagic capacity of cobalt chloride, an iron competing agent, and discover its neuroprotective effect at sub-lethal doses against hypoxia- or iron depletion-induced cell death in mammalian cells. Moreover, cobalt chloride supplementation extends lifespan and sustains locomotion in several nematode models of neurodegenerative diseases. Taken together, the authors suggest the beneficial effects of cobalt chloride across species via the activation of a hormetic response.In their research paper, In closing this Editorial, we would like to thank the authors and referees for their invaluable input in assembling this up-to-date Research Topic on the role of mitophagy in health and disease. We anticipate that the assortment of articles encompassed within this Research Topic will serve as a valuable resource and inspire additional research endeavors seeking to unravel the intricate role of mitophagy in the maintenance of cellular, tissue and organismal physiology.


Nanotechnology provides effective methods for precisely delivering chemotherapeutics to cancer cells, thereby improving efficacy and reducing off-target side effects. The targeted delivery of nanoscale chemotherapeutics is accomplished by two different approaches, namely the exploitation of leaky tumor vasculature (EPR effect) and the surface modification of nanoparticles (NPs) with various tumor-homing peptides, aptamers, oligonucleotides, and monoclonal antibodies (mAbs). Because of higher binding affinity and specificity, mAbs have received a lot of attention for the detection of selective cancer biomarkers and also for the treatment of various types of cancer. Antibody-conjugated nanoparticles (ACNPs) are an effective targeted therapy for the efficient delivery of chemotherapeutics specifically to the targeted cancer cells. ACNPs combine the benefits of NPs and mAbs to provide high drug loads at the tumor site with better selectivity and delivery efficiency. The mAbs on the NP surfaces recognize their specific receptors expressed on the target cells and release the chemotherapeutic agent in a controlled manner. Appropriately designed and synthesized ACNPs are essential to fully realize their therapeutic benefits. In blood stream, ACNPs instantly interact with biological molecules, and a protein corona is formed. Protein corona formation triggers an immune response and affects the targeting ability of the nanoformulation. In this review, we provide recent findings to highlight several antibody conjugation methods such as adsorption, covalent conjugation, and biotin–avidin interaction. This review also provides an overview of the many effects of the protein corona and the theranostic applications of ACNPs for the treatment of cancer. Off-target side effects, such as myelosuppression, mucositis, alopecia, organ dysfunction, and thrombocytopenia, are the most significant clinical challenge when using conventional chemotherapeutics . To imprHowever, the movement of NPs is hampered by biological barriers such as endothelial, cellular, skin, and mucosal barriers, which obstruct their targeting capabilities . ResearcThere are a number of approaches for achieving specific conjugation of antibodies on NP surfaces. The selection of the most suitable conjugation method is very important to preserve antigen binding ability. Improper antibody conjugation influences antigen binding affinity and specificity. Once injected into the body, the ACNPs face both physical and biological barriers , which eventually decrease the number of NPs at the target site . In blooAntibodies are Y-shaped glycoproteins produced by B-lymphocytes. These react specifically with antigens, which are responsible for the production or induction of specific antibodies . The speCancer is a highly heterogeneous condition that arises from several mutations in transforming and tumor suppressor genes. High rates of metastasis, invasion, relapse, and drug resistance are the main causes of treatment failures in cancer . TherefoSurface functionalization of NPs through conjugation of functional groups with biomolecules is one method to enhance the targeting efficiency . For effAdditionally, the functionalization also alters the surface composition and morphology of the NPs. The nature of interactions between NPs and antibodies is directly related to the type of functional groups present on the NPs surface and the surface charge –37. SurfAdsorption of antibodies on the surface of NPs is a non-covalent reversible binding method, which includes physical adsorption and electrostatic binding  38]. Ph. Ph38]. Ionic binding, in contrast, involves an interaction between surface of the antibody and surface of the NP, which are oppositely charged . This meCovalent binding of antibodies can be achieved either by adding functional groups on the NP surfaces or by chemical modification of antibodies. Covalent attachment provides high stability, prominent reproducibility, and strong interaction; therefore, changes of pH or ionic strength do not affect the interaction between antibodies and NP surfaces –46. The Carbodiimide chemistry: Carbodiimide conjugation is the commonly used coupling method for covalent binding of antibodies on NP surfaces by primary amine groups. Amine groups are abundant on the antibody surfaces and are highly susceptible for any reaction without chemical modification towards the various functional groups present on NP surfaces, such as aldehyde, carboxylic acid, and epoxide groups .–59.Clickficiency .These covalent conjugation methods are highly stable but associated with some drawbacks. These include aggregation and polymerization, cross-linking at multiple sites on the antibodies, the need for additional purification steps for the removal of linkers and catalytic agents, which results in low yield and poor reproducibility, and the random orientation of antibodies on the NP surfaces, resulting in a low accessibility of antigen binding sites .N-acetyl glucosamine and four mannose units. The four sugar moieties of avidin bind to biotin with high affinity [Non-covalent conjugation via adapter molecules ensures the availability of the Fab region of antibodies through the oriented immobilization via the Fc region. The binding of antibodies on nanoparticle surfaces is much stronger, but it is a reversible binding. Biotin–avidin interaction is the most commonly used binding strategy with adapter molecules . It reliaffinity . Additioaffinity . To avoiaffinity ,64.Another method to achieve oriented immobilization of antibodies is based upon the use of Fc binding proteins (such as protein-G and protein-A), which have ability to bind precisely with the Fc region of antibodies . WartlicThe covalent binding of amine groups of antibodies with carboxyl groups on the NP surfaces through carbodiimide conjugation is commonly accepted because it does not require a modification of the antibodies. After the conjugation of specific antibodies, the quantification of NP–antibody binding efficacy is an important factor to improve therapeutic efficacy. Researchers are actively working on quantification methods that are specific, rapid, sensitive, simple, and easy to use. The Lowry assay, UV spectroscopy, Bradford assay, and bicinchoninic acid assay are widely used and accepted methods to quantify the concentration of antibodies on NP surfaces in laboratories . Yet, thThe binding efficacy of antibody-conjugated NPs to overexpressed receptors on target cells is high because of the specific nature of the targeting antibody. ACNPs are expected to accumulate at the target site and to improve the tumor uptake. ACNPs receptor binding efficacies were determined using in vitro studies including cellular uptake and internalization studies .The high surface-to-volume ratio of NPs offers the advantage to manipulate their dimensions and functionalize their surface with multivalent targeting moieties to facilitate targeted delivery in tumor cells . NPs areThe small hydrodynamic diameter and unique structure of NPs offer increased adsorption and catalytic efficiency. The NP surface potential plays an essential role in the interaction between protein and NPs as opposite charges produce strong interaction and result in high conformational changes . The conProtein corona formation on antibody-conjugated NPs can have both negative and beneficial effects. Nayak et al. adsorbed bovine lactoferrin (BLf) onto AgNPs and studied the effect of this protein corona. They observed a higher internalization of BLf-AgNPs than of bare AgNPs, which indicates that the protein corona improved the intracellular efficiency of the NPs. Moreover, the BLf corona also increased the bioavailability of the NPs in THP1 cells and resulted in enhanced humoral immune response. The protein corona formation on Ab-conjugated NPs mediates a more specific and sensitive antibody–antigen interaction . The groThe protein corona formation results in the reduction or elimination of NP targeting capability by shielding or completely covering relevant functional groups. To block the adhesion of corona proteins on NP surfaces, various strategies have been established using surface barrier layers, such as polymer, protein, or biomimetic coatings, with the ultimate aim to prolong the blood circulation time of NPs. However, some of these strategies inhibit the internalization of NPs by cancer cells, resulting in limited therapeutic effectiveness. Accumulating evidence suggests that protein-repellent coating compounds (zwitterionic compounds) on NP surfaces prevent or minimize the corona formation. Precoating of NPs with specific proteins that increase the adsorption of plasma proteins with intrinsic targeting capacities also improve the targeting ability of the NPs. Moreover, researchers demonstrated that attachment of targeting moieties on the surface of corona-coated NPs increases the targeting capacity. These strategies are useful to shield the effect of protein corona formation and, therefore, improve the biodistribution profile and targeting efficacies of the NPs .High metastasis rates, drug resistance, and tumor relapse are the leading challenges in cancer diagnosis and treatment. The commonly used methods for the diagnosis of cancer involve identification of cancer-causing features in cells, such as DNA and RNA mutations, impaired expression of proteins, and changes in confirmation and cell morphology . These mIn some cases, ACNPs provide opportunities for image-guided therapy with overall theranostic applications. The NP-mediated targeted delivery ultimately provides advanced diagnostic and therapeutic options for early diagnosis and treatment of invasive and metastatic cancers.Based on these findings, it can be suggested that the ACNPs offer significant advancements in cancer diagnosis and treatment. ACNPs are, therefore, efficacious targeted agents with great theranostic ability, enhancing the overall quality of life in preclinical studies.Along with enormous progress in preclinical studies including improved intratumor drug delivery, enhanced therapeutic efficacy, and controlled release of chemotherapeutics at tumor sites, researchers evaluated the therapeutic potential of ACNPs in clinical trials. A literature study demonstrated that 13 targeted NPs had been progressed into clinical trials in 2013; however, their therapeutic efficacy in humans has not been proven yet . The avaIn cancer therapy, ACNPs have several advantages related to their ability to accumulate at tumor sites. More importantly, surface modification and functionalization of NPs to increase their therapeutic efficacy can be achieved very easily. The recent advancement in targeted delivery introduces the development of surface-modified nanosystems that can be used in screening, detection, and eradication of cancer cells and biomarkers, with great potential in theranostic applications. Despite these advantages, the design and fabrication of targeted NPs for cancer therapy is still very challenging regarding biocompatibility, pharmacokinetics, in vivo targeting efficacy, and cost-effectiveness. The optimization of these variables depends on NP design parameters such as size, shape, charge, composition, preparation method, and surface decorating moiety. The most crucial aspect in the future development of ACNPs will be the design and fabrication of multiple targeting moieties with the ultimate goal of diagnosis and treatment with better efficacy. However, ACNPs have proven their ability to deliver chemotherapeutics specifically to the tumors and are currently the most beneficial targeted conjugated therapy in preclinical studies. However, ACNPs have not been passed phase-III clinical trials yet. It is still very important that researchers understand better the chemistry between NPs and antibodies to develop improved ACNPs. Current knowledge of targeted delivery, tumor vasculature, and interactions of ACNPs in the human body is very limited and, hence, results in failure in clinical trials. To completely exploit the advantages of ACNPs in both preclinical and clinical studies, it is substantial to analyze the gaps between formulation development, selection of appropriate animal models, and clinical safety in humans. Additionally, early detection is one of the most important issues in cancer treatment, as this improves survival rates by approximately five years and also lowers the overall treatment cost. However, it is critical that diagnosis and treatment are extremely accurate, otherwise this would result in misdiagnosis and overtreatment. Unfortunately, the success rate for clinical studies is also very low because of the lack of efficacy. Hence, more advancements are required in the area of targeted delivery to benefit from the advantages of ACNPs in cancer treatment.ACNPs are one of the emerging targeted delivery systems. They combine the advantages of NPs and antibodies, such as increased surface to volume-ratio, surface modification and functionalization, and improved cellular uptake and intracellular stability. In cancer chemotherapy, ACNPs have shown immense potential to achieve targeted drug delivery and combination therapy, to overcome drug resistance, to reduce toxicity, to enhance immune response, and to monitor treatment response through theranostic applications. There are numerous preclinical studies demonstrating the above benefits of ACNPs in cancer treatment; however, these advances are not yet clinically transformable because of issues such as lack of tumor specificity and selectivity, antibody selection issues, immune reactions against ACNPs, stability of ACNPs, heterogeneity of tumors, regulatory approvals, scale-up and production, and cost considerations.