The most common malignancies in childhood are leukaemias, brain tumours, lymphomas, neuroblastomas, soft tissue sarcomas and kidney tumours. At present, about 80% of childhood cancers can be treated successfully, which has significantly increased long-term survival. Concomitantly, adult gonadal function in childhood cancer survivors has become a concern. However, the immediate effect of cancer and its management on the reproductive axis function has received less attention. We conducted a review of the effects of malignancies and their treatments on the gonadal axis during childhood and adolescence. Some results are controversial, probably because the analyses do not distinguish between the malignancy types, their treatments and/or the age at treatment. However, there is agreement that cancer can partially affect gonadal function before treatment, as revealed by low circulating levels of inhibin B and anti-Müllerian hormone. Subsequently, chemotherapy transiently impairs the somatic component of the gonads (i.e. testicular Sertoli cells and ovarian granulosa cells) with normalization after treatment ends. The impact of chemotherapy may persist through adulthood after more intensive chemotherapy regimens, radiotherapy and conditioning for haematopoietic stem cell transplantation, when there is a severe impairment of the somatic component of the gonads or of the stem germ cells. However, little attention has been driven to the immediate effects of cancer and its treatment observed during childhood, especially their impact on the somatic cell populations of the gonads, which are the most numerous cell types before pubertal development. In this narrative review, we discuss the existing evidence on the immediate effects of cancer and its treatment on the function of the gonadal axis during childhood and adolescence.The development of effective therapies for the management of cancer in childhood has led to outstanding improvements in survival over the past decades. With this, a growing population of childhood cancer survivors are subject to long-term sequelae impacting on health. Endocrine disorders, and especially reproductive function in adulthood, have become an important concern.5 Understanding the differences in the physiology of the HPG axis during postnatal development is essential for the interpretation of the clinical presentation of HPG disorders in children and adolescents.The most frequent concepts used to describe the physiology and nomenclature of the pathological conditions of the hypothalamic– pituitary–gonadal (HPG) axis derive from observations in the adult.6 Additionally, follicles produce inhibin B, whereas the corpus luteum produces inhibin A, which exerts negative feedback on pituitary FSH.7 Inhibin B production increases progressively as follicles grow from 4 to 12 mm.8 Conversely, anti-Müllerian hormone (AMH) is predominantly secreted by small follicles ≤2 mm, showing a progressive decrease in follicles 4–8 mm to reach very low production in follicles ≥10 mm. Serum AMH levels remain relatively stable during the menstrual cycle,11 reflecting the ovarian reserve12 and decline towards menopause.13In the adult woman of reproductive age, follicle-stimulating hormone (FSH) regulates the function of granulosa cells of the follicle while luteinizing hormone (LH) acts on theca cells and the corpus luteum. Androgens are synthesized by the theca in response to LH and aromatized to oestrogens in the granulosa cells in response to FSH.14 and a subsequent increase with one or two peaks during the first year of life.16 This active period of the female HPG axis declines after 1 year of age, when serum LH and oestrogens become undetectable, and serum FSH and inhibin B are detectable but low.16 AMH remains stable during childhood,17 reflecting the pool of small follicles that do not progress to full maturation.The newborn shows very low levels of gonadotropins and ovarian hormones at birth,20 Menarche does not indicate full maturation of the HPG axis, and menstrual cycles may be irregular during the first years in normal adolescents.22. Ovulatory cycles, as detected by adequate progesterone production by the corpus luteum, represent only 15% in the first post-menarchal year and progressively increase to approximately 40% in the third year and 75% in the sixth year.23With pubertal onset, between 8 and 13 years of age, gonadotropins and local factors lead to the activation of terminal follicular growth.6 The oocyte pool is fixed at birth, and new oogonia cannot be formed. The true ovarian reserve is represented by primordial follicles formed by primary oocytes and a single layer of flattened granulosa cells. During infancy and childhood, there is a non-cyclic recruitment of primordial follicles that progress to the primary follicle stage. These are formed by an arrested primary oocyte and proliferating granulosa cells in several layers; nonetheless, follicles do not progress beyond the medium (7–11 mm) antral stages.24 At puberty, germ cell division (meiosis) resumes, and granulosa and theca cells are also very active. Any insult to the oocyte triggering its apoptosis irremediably leads to follicular loss, whereas disorders primarily disrupting the somatic follicular cells may be reversible until oocyte survival is affected.At all ages, the interaction between germ cells and the somatic components of the ovarian follicles, i.e. granulosa and theca cells, is vital. Before puberty, the germ cell population is composed of primary oocytes arrested in the first meiotic division.26 A concerted action of androgens and FSH is necessary for normal sperm output.In the adult male, LH drives Leydig cell testosterone secretion, whereas FSH regulates Sertoli cell function and inhibin B production. Androgens and inhibin B exert negative feedback on LH and FSH secretion, respectively.14 However, high gonadotropin and androgen levels persist for only the first 3–6 months.28 Curiously, although these hormone levels are in the same range as those observed in adults, the testes remain immature and sperm production does not occur, due to the lack of expression of the androgen receptor in Sertoli cells.29 During the rest of infancy and childhood, most of the HPG axis becomes quiescent as in the female. Gonadotropin levels decline: LH is very low or undetectable, resulting in a complete disappearance of functional Leydig cells and androgen synthesis. Sertoli cells remain immature but active, and their function can be detected through the determination of AMH and inhibin B in serum.30Similarly to females, the newborn males show very low levels of gonadotropins and testicular hormones at birth, which increase progressively during the first month of life.31With the onset of puberty, between 9 and 14 years of age, FSH initially boosts Sertoli cell proliferation, which provokes testicular enlargement beyond a volume of 4 mL. Within the testes, testosterone induces Sertoli cell maturation and adult spermatogenesis. Clinically, this is characterized by a decline in AMH and an increase in inhibin B, together with a progressive increase in testicular volume to 15–25 mL.The dynamics of germ and somatic cell populations of the testis differ from that of the ovary. As mentioned, Leydig cells are present in the first 3–6 months of life, then they are no longer seen until the onset of puberty. Immature Sertoli cells have proliferating capacity in response to FSH until intratesticular testosterone increases in puberty and provokes their maturation. The germ cell population is represented by spermatogonial stem cells, which can renew throughout life. Spermatogonia proliferate by mitosis during the active period of the HPG axis after birth but do not enter meiosis. During childhood, the rate of spermatogonial mitotic divisions decreases and it boosts again after pubertal onset. By then, spermatogonia can also enter meiosis to form spermatocytes, spermatids and spermatozoa. Defective function of Sertoli cells severely compromises germ cell survival and proliferation. Defective androgen production by Leydig cells results in lack of progression through meiosis but does not necessarily affect the survival of spermatogonia. Unlike the ovary, germ cell loss in the testis does not lead to seminiferous tubule or Leydig cell disappearance. Indeed, in the absence of germ cells, androgen production may be normal, and Sertoli cells can produce AMH normally. Nonetheless, inhibin B production is impaired as it needs both FSH and germ cell action on Sertoli cells.32 Hypogonadism has different clinical presentations and health consequences depending on the age at which it is established, the gonadal cell population initially impaired and the level of the HPG axis primarily affected (Table 1).From this discussion, it follows that the concept of hypogonadism needs to be adapted when applying it to the paediatric population. Based upon developmental physiology, hypogonadism can be defined as an impaired gonadal function, based on the patient’s age, which may result in a decreased secretion of sex steroids, AMH and/or inhibins, and/or a disrupted gamete production.33 and a similar pattern is observed in anorchid boys 36 and in boys with primary hypogonadism associated with cryptorchidism,37 indicating that approximately 35–75% of the patients would go undiagnosed if gonadotropin elevation was required to diagnose primary gonadal failure in patients of prepubertal age.38An important concept in prepubertal patients is that, due to the central inactivation of the HPG axis, primary hypogonadism usually presents with normal gonadotropin levels . Indeed, girls with complete gonadal dysgenesis due to Turner syndrome have normal FSH and LH levels between ages 4 and 10 years,41The activity of the HPG axis is clinically inapparent in the female during foetal life and in childhood. Actually, girls with congenital primary hypogonadism  or with central hypogonadism are most usually diagnosed only at the age of puberty due to the absence of thelarche and menarche.44 Low levels indicate a decreased follicular pool and undetectable levels denote the absence of functional ovarian tissue. Noteworthy, the definitions of POI or POF coined for the adult woman, based on the finding of serum concentrations of FSH >25 or 40 IU/L twice at least 1 month apart, low sex steroid levels and amenorrhoea for at least 4 months in a female aged less than 40 years,45 cannot be applied in paediatrics: occurrence of low sex steroids and no menstrual cycles reflects the physiological state in prepubertal girls and FSH remains below 25–40 IU/L during most of childhood in girls with primary hypogonadism.As discussed earlier, except for the transient activation period of the gonadal axis in the first year after birth, serum determination of oestradiol, progesterone and gonadotropins gives little or no information. Conversely, since small ovarian follicles secrete AMH and inhibin B, these biomarkers may be used throughout childhood to assess the status of the female gonads.46 and for persistent penile enlargement during the first 3–6 months of postnatal life,47 micropenis and cryptorchidism are suggestive of congenital hypogonadism in the male newborn.48 Conversely, hypogonadism acquired later in childhood may go clinically undiagnosed. Circulating sex steroid and gonadotropin levels are not helpful, and only low serum levels of AMH50 or inhibin B52 may be indicative of gonadal function impairment.Unlike the observations in females, in males the function of the HPG axis during foetal and early postnatal life has a clinical impact. Because androgens are essential for testicular descent and penile growth during intrauterine development,53 The epidemiology of cancer in children and adolescents differs significantly from that observed in adults. Indeed, in the paediatric population, the most common types of cancers include leukaemias, brain tumours, lymphomas, neuroblastoma, soft tissue sarcomas and kidney cancer, especially Wilms tumours, with differences according to age and world regions. In high-income countries, cancer is the second leading cause of death in children, after accidents. The overall annual incidence of cancer is approximately 141 per million under the age of 14 years and 185 per million in adolescents aged 15–19 years. There is a slightly higher incidence rate in boys than in girls, with an overall incidence sex ratio of 1.14. However, sex-specific incidence varies according to cancer type, and renal, gonadal and epithelial tumours are more frequent in girls.Epidemiology of cancer in children and adolescents Each year, approximately 400,000 children and adolescents below the age of 19 years are diagnosed with cancer worldwide.Table 1).53 Tumours of the central nervous system follow in frequency with 20%, lymphomas account for ∼11%, neuroblastomas represent 7%, soft tissue sarcomas and renal tumours ∼6% each, bone tumours 4%, and germ cell/gonadal tumours, epithelial tumours/melanomas and retinoblastomas account for 3–4% each.In children below the age of 14 years, leukaemias represent approximately 33% of all cancer types (Table 1), lymphomas are the most common tumour type, with approximately 23% of all cancers, followed by epithelial tumours/melanomas (21%), leukaemias (15%), germ cell/ gonadal tumours (12%), central nervous system tumours (11%), and bone and soft tissue sarcomas (between 7% and 8% each).53In adolescents aged 15–19 years , acute myeloid leukaemia (AML) and non-Hodgkin lymphoma (NHL). Successful treatment currently occurs in 90% of cases, based on the use of intensive multi-drug, induction and consolidation regimens.57Hematological malignancies represent approximately 40% of cancers in the paediatric population,ClinicalTrials.gov Identifier: NCT00764907) .58 There are three risk groups: standard, intermediate and high risk. These are based on blast response to prednisone and intrathecal methotrexate, patient age, initial white blood cell count, percentage of blasts in bone marrow, and the presence of one of two chromosomal rearrangements: the t translocation resulting in the fusion of the ABL proto-oncogene located on chromosome 9q34 with the breakpoint cluster region (BCR) located on 22q11.2 and giving rise to the formation of the Philadelphia chromosome (BCR-ABL); or the t translocation where the mixed-lineage leukaemia (MLL) gene on chromosome 11q23 fuses with the AF4 gene in chromosome 4q21 producing a chimeric MLL/ AF4 fusion transcript.In the case of ALL, risk stratification and treatment are usually assigned following the criteria set by the ALL IC-BFM 2002 trial .60 Patients with T cell ALL and high-risk patients aged 1 year or more usually receive 12 Gy prophylactic cranial radiotherapy, whereas children with initial involvement of the central nervous system receive therapeutic cranial radiotherapy with age adjustment: 12 Gy between 1 and 2 years old and 18 Gy for those older than 2 years.58 Gonadal irradiation is required only in cases of leukaemic gonadal relapse.Standard- and intermediate-risk ALL and AML usually respond to less aggressive chemotherapy and do not require preconditioning chemotherapy for haematopoietic stem cell transplantation (HSCT) or HSCT conditioned with total body irradiation (TBI).55 Tumours of the sympathetic nervous system are treated with spinal irradiation, which may affect the hypothalamic–pituitary region or the gonads, depending on the location of the tumour.Brain tumours are treated with surgery and may need radiation therapy. The tumour itself or the surgical procedure may affect the hypothalamic– pituitary region. The radiation dose impairs endocrine function variably: growth hormone deficiency and precocious puberty can be seen with cranial radiotherapy doses ≥18 Gy, whereas other endocrine axis manifestations require doses ≥30 Gy.60Soft tissue sarcomas stage I, renal (Wilms) and germ cell tumours may only require surgery. When no radiotherapy is required, the risk of endocrine compromise is low. Conversely, the risk increases in the cases of metastatic or high-risk sarcomas requiring high cumulative doses of alkylating agents  and testicular irradiation.1 Because cancer itself, as well as chemotherapy and radiotherapy, can affect gonadal function, loss of fertility and sex hormone deficiencies have become important concerns for childhood cancer survivors. We have conducted a review on the immediate effect of the occurrence of malignancies and of their treatment on gonadal function during childhood and adolescence.The notorious advances made in the diagnosis and management of malignancies during childhood and adolescence has led to a growing population of cancer survivors who are susceptible to long-term sequelae impacting on health issues, especially endocrine disorders.Table 2). Cell-cycle-specific agents inhibit mitosis by impairing DNA synthesis, thus affecting cells with a high proliferation rate. Germ cells involved in oogenesis and spermatogenesis during pubertal development are the most susceptible cell populations to cell-cycle-specific agents.62 Nonetheless, granulosa cells of small ovarian follicles and testicular spermatogonia and Sertoli cells also have a moderate proliferating activity in childhood. These cell types could also be affected by cell-cycle-specific drugs. The difference in their recovery is that the oocyte pool is fixed, with no stem cell renewal after birth, while granulosa cells, spermatogonia and Sertoli cells can renew if the gonads are not completely depleted of them by treatment. Another point to consider is that oocyte depletion results in granulosa cell exhaustion in the ovary, whereas spermatogonial depletion does not overtly affect Sertoli or Leydig cell numbers in the testis. On the other hand, non-cell-cycle-specific agents provoke direct DNA and RNA damage in both proliferating and cell-cycle-arrested cells, inducing apoptosis.61 Therefore, all gonadal cell types at any age would be susceptible to these agents.Two main characteristics of the developmental biology of the HPG axis need to be considered when analysing the potential effects of childhood malignancies and their treatments on gonadal function. One is the different mitotic activity of germ and somatic cells of the gonads between childhood and puberty. The other is the difference in the activities of the germ cells and the somatic cells between the ovary and the testis. All this may explain the differential effects of the diverse chemotherapies and radiotherapies on the HPG in childhood and adolescence. According to their mechanism of action, chemotherapy agents can be classified into ‘cell-cycle-specific’ and ‘non-cell-cycle-specific’ drugs , until several months or years after treatment end, and those still of paediatric ages.69 One cross-sectional study focused on the ovarian status at the time of diagnosis,70 whereas two other studies evaluated the effect of cancer and its treatment only after the end of therapy.72Initially, results from studies carried out with cohorts of adult survivors of childhood cancer drove the attention to the long-term deleterious effects of chemotherapy or radiotherapy on reproductive function.70 Brougham et al67 were the first to report in 22 girls  with solid malignancies requiring treatment with pulsed chemotherapy or radiotherapy that serum AMH was slightly but not significantly below that of the reference population mean. The analysis – which did not discriminate between tumour type or consider the duration of treatment (5–39 months) or the length of follow-up (4–43 months) - found that serum AMH was less affected in older girls, declined significantly during the initial six treatment cycles, and recovered to pre-treatment values in girls after >12 months of the end of treatment. Serum inhibin B was also below the mean at diagnosis and declined significantly in the first 4 cycles of oncological treatment. Although its circulating levels did not recover during followup, serum FSH did not increase. This could be due to the fact that most of the girls were still younger than 9 years. As already mentioned, it has been known for a long time that primary ovarian insufficiency can present with normal FSH in girls of prepubertal age.33Results may seem somewhat controversial regarding certain endpoints, and this could be due to the fact that some studies do not distinguish in their analyses the potential effects of the different underlying malignancies, their treatments and/or the age  at which treatment was initiated and finished. However, most studies conclude that ovarian function is mildly impaired at the time of diagnosis, before any treatment is initiated.70 in 114 girls aged <18 years with haematological malignancies and 94 with solid tumours clearly showed that serum AMH is reduced at the time of diagnosis, in correlation with impairment of general health status in girls. The authors suggest that other factors, besides small follicle number, may influence ovarian AMH secretion.The study by van Dorp et al66 could not detect any impairment in ovarian function at diagnosis but showed a decline of serum inhibin B to undetectable levels during chemotherapy, with a variable degree of recovery 1–6 months after the end of treatment. In agreement with the study by Brougham et al.,67 Crofton et al did not find any significant changes in gonadotropin levels.In a small series of nine prepubertal girls with haematological and solid malignancies, Crofton et al.69 assessed at diagnosis 129 girls aged <18 years with cancer, of whom 49 were followed during and after treatment. The analysis did not distinguish between haematological (n=31) and solid (n=18) malignancies. Serum levels of AMH were in the lowest part of the reference range in both premenarcheal and postmenarcheal girls at diagnosis. In premenarcheal girls undergoing standard-risk chemotherapy, no significant changes were seen in serum AMH during treatment and an increase was found during follow-up ≥12 months after treatment end. Conversely, serum AMH declined and did not recover to pretreatment levels in girls receiving high-risk chemotherapy. In postmenarcheal patients, serum AMH had already decreased significantly 1 month after treatment start and remained low after treatment end. The authors concluded that gonadotoxicity occurs more frequently in postmenarcheal girls and that premenarcheal girls are more likely to recover the function of the HPG axis. Therefore, they hypothesize that preventive treatment with gonadotropin hormone-releasing hormone analogues is useful to preserve ovarian function.In a more recent and larger study, van der Kooi et al.73 However, it is extremely infrequent nowadays, as revealed by an immunohistochemical assessment performed in a series of cryopreserved ovarian cortex samples obtained for fertility preservation before chemotherapy or radiotherapy from patients with leukaemia. Nevertheless, in approximately 75% of the cases, assessment of leukaemia genetic biomarkers in the ovarian tissue by polymerase chain reaction suggested the existence of leukaemic cells in the cryopreserved samples.74 Although their viability and malignancy is uncertain, the reimplantation of cryopreserved ovarian tissue cannot be recommended in patients with leukaemia.Gonadal infiltration is one possibility to explain primary hypogonadism (ovarian dysfunction) before the start of treatment. The incidence of gonadal infiltration was high several decades ago.72 assessed ovarian function in girls who underwent HSCT. The study included six girls 6–11 years old with ALL, who received allo-HSCT, and six girls 5 months to 13 years old with neuroblastoma, Hodgkin disease or NHL, treated with auto-HSCT. In all cases, the ovarian reserve was severely impaired, as reflected by extremely low or undetectable serum AMH between 6 months and 11 years after HSCT. However, the HPG axis still showed partially or completely preserved function in six cases.High-risk treatments include those requiring HSCT. In a recent study, Wędrychowicz et al71 found that 10 patients developed early puberty in association with low-dose cranial radiotherapy and required treatment with a gonadotropin hormone-releasing hormone analogue. After followup between approximately 6 and 14 years, clinical progression of puberty was normal in 15 girls; FSH and LH were in the reference range in 14 of them and high in 1, indicating an incipient primary ovarian failure. Serum AMH and inhibin B were normal in 11 cases and low in 4, reflecting a decreased ovarian reserve that did not yet have an overt impact on the HPG axis function. On the other hand, abnormal progression of puberty was seen in 7 girls: 2 with central hypogonadism, 4 with primary hypogonadism and 1 with combined  hypogonadism.In 22 patients with medulloblastoma or ependymoma aged <11 years, treated with surgical resection, cranial and/or spinal irradiation and chemotherapy, Cuny et al.75 In pubertal girls, the impact of chemotherapy could be longer lasting, as oocytes have reinitiated meiotic divisions. At any age, more intensive chemotherapy regimens, abdomino-pelvic radiotherapy and HSCT usually result in a severe and persistent damage of the ovarian tissue. Cranial radiotherapy affects the hypothalamic–pituitary axis: doses 18–30 Gy induce early HPG axis activation resulting in precocious puberty, whereas doses >30 Gy provoke gonadotropin deficiency, resulting in delayed/absent/arrested puberty.In summary, cancer partially affects ovarian function, probably by impairing the general health status. In certain malignancies, such as leukaemias, ovarian infiltration - even submicroscopic - may also explain a decline in AMH and inhibin B production. The underlying pathogenic mechanisms need to be unveiled. In most cases, chemotherapy during childhood impairs ovarian function transiently, as revealed by a decline in AMH and inhibin B levels, with a normalization after treatment end. This might be explained by an effect of cell-cycle-specific chemotherapy agents mainly on proliferating granulosa cells of small follicles without inducing an increased germ cell apoptosis (i.e. not affecting the ovarian reserve).63 Here, also, the conclusions obtained from studies carried out decades ago78 may be outdated, owing to the progress made in diagnosis and treatment regimens. Results of the most recent studies of large cohorts of boys with cancer focused on the function of the HPG axis are summarized in Table 4. Two longitudinal studies assessed testicular function from the time of diagnosis, before any treatment, until several months or years after the end of treatment,69 whereas the other two longitudinal studies evaluated the effect of cancer therapy on testicular germ cells once therapy was initiated, during and after treatment.80 Two cross-sectional studies focused on testicular function at diagnosis.82 Finally, two studies analysed the effect of cancer and its treatment exclusively several months or years after treatment end, yet still in the paediatric age group.83As in females, the first evidence of the deleterious effects of childhood cancer treatment on male reproductive function came from studies in cohorts of adult survivors.66 could not detect any impairment in testicular function at diagnosis, during or after treatment, except for one case who received an intensive chemotherapy regimen. However, much larger studies yielded somewhat different results. Krawczuk-Rybak et al81 found that testicular volume and serum inhibin B were normal at diagnosis in 48 prepubertal boys - not distinguishing between those with haematological (n=34) and those with solid (n=14) malignancies - but decreased in 36 pubertal patients with either haematological (n=16) or solid (n=20) cancer with no impact on gonadotropins or testosterone. On the other hand, in a large crosssectional study, Wigny et al82 found normal inhibin B levels at diagnosis only in nine boys with nephroblastoma but a significantly lower mean inhibin B in serum in 215 boys with other solid tumours or haematological malignancies, not distinguishing between prepubertal and pubertal status. Moreover, the recent study by van der Kooi et al.69 found that serum inhibin B could be normal or low before treatment start in both prepubertal and pubertal boys with solid (n=19) or haematological (n=40) cancer and that it further decreased during the first month of therapy, with a partial recovery 10 months after the end of chemotherapy, which could be associated with cranial, chest and/or abdominal radiotherapy. Boys treated after pubertal onset seemed to be more affected than those treated at a prepubertal stage.In a series of 16 prepubertal boys with haematological and solid malignancies, Crofton et al.83 found that testicular volume was not affected in prepubertal patients but was reduced in pubertal ones; mean serum inhibin B was significantly lower independently of pubertal status, while gonadotropins and testosterone were within the normal range. Somewhat different results were reported recently by Grinspon et al.59 in a large series of boys with leukaemia or NHL 1–12 years after the end of chemotherapy, associated with cranial radiotherapy or HSCT, in certain cases. While testis volume was normal in boys treated before puberty, it could be affected in those treated during puberty . These results suggest that the chemotherapy regimens used, including drugs of low risk for spermatogenic toxicity and doses of alkylating agents that are below the expected toxicity risk, do not significantly affect the Sertoli cell population, which accounts for the major proportion of testicular volume in childhood. Serum AMH and testosterone were within the normal range in all cases, while gonadotropins were normal in those treated before puberty and normal or slightly elevated in those who received treatment until after pubertal onset, especially in those who received high-risk therapy . Altogether, these observations indicate that the reduced testicular volume observed during puberty is due to the impairment of the germ cell population with no major damage of the somatic cell populations of the testis.Analysing, retrospectively, boys and adolescents who received chemotherapy - with associated cranial radiotherapy in certain cases - for leukaemia several years earlier, Krawczuk-Rybak et al.79 conducted a longitudinal study in boys with ALL. Compared with the end of induction therapy, the number of spermatogonia significantly decreased by the end of chemotherapy in boys who had received high-risk treatment including cyclophosphamide. Without cyclophosphamide, no significant effect on the number of spermatogonia was observed, but the initiation of spermatogenesis was delayed. In a more recent survey, including prepubertal boys with ALL treated with chemotherapy and cranial or testicular radiotherapy, Poganitsch-Korhonen et al.80 found that while the mean number of spermatogonia decreased in boys treated with alkylating agents compared with non-alkylating drugs, a complete depletion of spermatogonia is rare, thus allowing for a recovery of spermatogenesis during puberty in most cases.The two studies that focused on the germ cell population confirmed the already known toxicity of alkylating agents. Nurmio et al.In summary, Sertoli cell endocrine function of the testis may be affected by cancer during childhood and adolescence at the time of diagnosis, similarly to what is observed in ovarian endocrine function in girls. In boys, this is reflected by inhibin B levels in the low/normal range or clearly below reference levels. Chemotherapy acutely impairs Sertoli cell function, but it does not seem to affect Sertoli cell numbers, as can be deduced by the recovery of inhibin B or AMH levels in the follow-up at least 1 year after the end of treatment. Leydig cell function does not seem to be affected, as revealed by normal testosterone production during puberty. However, the slight increase in gonadotropins observed in some cases may be the reflection of a mild compromise of Sertoli and Leydig cell function. Non-cellcycle-specific drugs, such as alkylating agents, transiently affect spermatogonial stem cell numbers, although they rarely produce a complete depletion. Consequently, azoospermia is not a frequent consequence of standard therapy for haematological malignancies. Intensive (high-risk) treatments including conditioning for HSCT and testicular radiotherapy are more deleterious, and pubertal status at the time of treatment is associated with a more severe effect on the HPG axis.Compared with the extensive experience of the long-term effects of treatment on the reproductive axis in adult childhood cancer survivors, there are limited studies that have addressed the immediate effects of cancer itself and its treatment on gonadal function during childhood. In some cases, contradictory results may arise from studies not analysing separately the effects of malignancy types, treatments using drugs with different mechanisms of actions, or the pubertal status when treatment was administered. There is consensus that gonadal function may be partially affected at the time of diagnosis, before treatment is initiated, and that standard-risk chemotherapy has a transient deleterious effect on the somatic component of the gonads (i.e. Sertoli cells of the testis and granulosa cells of the ovary). The germ cell population is rarely completely depleted, which explains why gametogenesis is frequently present in young adult survivors. This raises the question of whether the concept of ‘gonadotoxic’ used for cancer treatments in adulthood should be applied identically in childhood.Unfortunately, no established protocols exist for the assessment of the gonadal axis during the follow-up of children with cancer. Based on the concepts of the pathophysiology of hypogonadism in paediatric age groups discussed in this review and of the scarce published findings on the effects of cancer and its treatment on the gonadal axis during childhood, the clinical assessment that may be suggested for paediatric patients certainly differs from the well-established protocols used in adults. In boys, direct assessment of the germ cell population is not feasible, and the evaluation of the LH–Leydig cell axis would require stimulation tests that may not be informative. Therefore, measurement of direct serum markers of Sertoli cell function (i.e. inhibin B and AMH) seem to be the most sensible decision. Measurement of serum FSH, as an indirect marker of Sertoli cell dysfunction, may not be informative before pubertal onset. Similar concepts are applicable to the followup of prepubertal girls: serum AMH and inhibin B seem to be the most reliable markers of the function of the hypothalamic–pituitary–ovarian axis, while FSH, LH and sex steroids give little information. For pubertal girls, the reestablishment of menstrual cycles with normal levels of gonadotropins and sex steroids indicates the recovery of a functional axis, but does not necessarily predict a normal status of the ovarian follicle reserve, which may be estimated by measuring serum AMH. In summary, further research is warranted to clarify the extent and duration of the specific effect of the different chemotherapy regimens on each gonadal compartment.


Humans have always been exposed to tiny particles via dust storms, volcanic ash, and other natural processes, and our bodily systems are well adapted to protect us from these potentially harmful external agents. However, technological advancement has dramatically increased the production of nanometer-sized particles or nanoparticles (NPs), and many epidemiological studies have confirmed a correlation between NP exposure and the onset of cardiovascular diseases and various cancers. Among the adverse effects on human health, in recent years, potential hazards of nanomaterials on female reproductive organs have received increasing concern. Several animal and human studies have shown that NPs can translocate to the ovary, uterus, and placenta, thus negatively impacting female reproductive potential and fetal health. However, NPs are increasingly being used for therapeutic purposes as tools capable of modifying the natural history of degenerative diseases. Here we briefly summarize the toxic effects of few but widely diffused NPs on female fertility and also the use of nanotechnologies as a new molecular approach for either specific pathological conditions, such as ovarian cancer and infertility, or the cryopreservation of gametes and embryos. In European legislation, a nanomaterial is defined as “an insoluble or bio-persistent and intentionally manufactured material with one or more external dimensions, or an internal structure, on the scale from 1 to ≤100 nm” . The mosNP spread in the environment is very relevant. Indeed, we can distinguish natural nanoparticles from those man-made and then dispersed ,7. AtmosOther NPs gaining significance as a pollutant are nanoplastics . The toxDepending on its composition, an NP can be lipophilic or hydrophilic: the former is able to cross cell membranes by diffusion, while the latter needs active transport . NPs canAfter entering in the circulatory system via various items , they can exert their negative toxic effects on cells by increasing the production of reactive oxygen species (ROS), damaging DNA and mitochondria and even inducing cell death . Table 12, CuO, CeO2 and FeO. Among these, TiO2 NPs are particularly popular in biomedical applications, being employed for the fabrication of biomedical devices and for bioimaging [With regard to metallic NPs, their effects depend mainly on their composition, morphology, size and crystal structure. They are characterized by a metal core composed of inorganic metal or metal oxide usually covered with a shell formed by organic, inorganic material or metal oxide. The most widely used metallic NPs in medicine, food, agriculture and industry include Ag, Au, ZnO, TiOoimaging . Consideoimaging .2NPs as a food coloring and flavoring agent is no longer considered safe because of its effects on DNA. Despite its low bioavailability, it can accumulate in the body reaching high chronic concentrations especially in long-living animals and breeding animals [2NPs in cosmetics as a preservative and colorant has been reduced to 25% in masks and hair products, a value considered safe without prejudice to its carcinogenic capacity by inhalation [Since 2015, the European Commission has launched a series of proposals with the aim of reducing their use both to safeguard human health and the environment . In 2021 animals . To date animals ,58. The halation . Other NThe reproductive toxicity of these NPs is becoming a public health problem due to the use of NP-containing consumer products and occupational exposure. Both male and female reproductive organs are vulnerable, but in females the presence of NPs may compromise fertility and eventual fetal development more dramatically due to the fixed and not renewable number of oocytes present at birth in the mammalian ovaries. Furthermore, NP accumulation seems to be strongly conditioned by menstrual cycle, as demonstrated by the 2- fold increase in ovarian and uterine NP content occurring during mouse ovulation in comparison with the other stages of estrus cycle . In thisSince NP intake via skin is considered to be below detection level, ingestion and inhalation represent the major route by which NPs are transferred to organs and cells, as confirmed by their presence in the circulatory and lymphatic systems as well as urine . Since N2 is produced in three commercial forms named rutile, brookite and anatase, which is the most utilized [Both NPs are commonly used in a wide range of products, from paints to cosmetics. nTiOutilized . In rece2NPs can damage ovarian cells have mainly been studied in laboratory animals. Experimental data demonstrated that a long-term administration (5–8 weeks) of TiO2NPs (1–10 mg/kg/d) to female mice caused alterations of gonadotropin and sex steroid release, cysts formation and follicular atresia together with significant reduction of pregnancy rate due to extensive ovarian inflammation caused by elevated ROS production [2NPs (1.25–2.5–5 mg/kg for 90 consecutive days) in both the cytoplasm and nuclei of follicle cells [2NPs [2 (2.5–5–10 mg/kg for 30 days), but differences can be ascribed to different treatment times and different mouse strains used in the experiments.The mechanisms by which TiOoduction ,66,67. Ioduction . Ovarianle cells . The risls [2NPs . Contrasls [2NPs  and Zhaols [2NPs  followinThe rapidly expanding production and use of ZnONPs in consumer products  also stimulated studies on this nanomaterial and its ability to alter/disrupt cell functions. nZnO toxicity seems to mainly be due to Zn ion release, although an intrinsic particle toxicity should be considered especially after long-term exposure . In fema2NPs inhibited rat follicle development and oocyte maturation in a size- and concentration-dependent manner [2 at increasing concentrations (12.5–25–50 μg/mL), follicle development and oocyte maturation were dramatically inhibited only in the presence of 25 nm TiO2 at 25 μg/mL or above. The presence of TiO2NPs did not hamper the process of cumulus expansion in mice, while a dramatic negative effect was caused by ZnO NM-110 and NM-111 at relatively low concentrations (1 mg/mL). Indeed, experimental data showed that ZnONPs, but not SiO2-coated ZnONPs, affected the expression of genes fundamental for the regulation of this process. The negative effect seems to be based either onion release or NP presence per se [The presence during culture of TiOt manner . In facte per se .2NPs (5–50 μg/mL for 96 h), the negative impacts on somatic and germ cells were significantly different. First of all, TEM analysis showed the absence of ZnONPs in antral follicular cells, while TiO2 NPs were easily detectable in theca, granulosa and cumulus cell cytoplasm as agglomerates enclosed in small vesicles or near mitochondria. Moreover, the presence of invaginations on cell surface confirms that TiO2NPs were internalized by micropinocytosis or clathrin-mediated endocytosis. Both NPs had a great impact on cytoskeleton arrangement and trans-zonal projections (TZP) between somatic cells and oocytes, which play a central role in the fine regulation of normal oocyte and follicle development [2: increase; ZnO: decrease), but none of them have been found in cultured oocytes that were indirectly damaged because of TZP disruption. Moreover, nZnO toxicity, evidenced by ROS increase, was higher than that caused by nTiO2, probably as a result of Zn ion release in culture media. A similar mechanism has been described as occurring also in other cells [2 and nAl2O3 were evaluated on Chinese hamster ovarian (CHO-K1) cells. Changes in lysosomal and mitochondrial dehydrogenase activity as well as the formation of perinuclear vesicles in CHO-K1 cells were demonstrated after treatment with both NPs, but none was detectable in the nuclei [Another recent study performed on mouse antral follicles  showed telopment . Surpriser cells . The cyte nuclei .Other metallic NPs can impair follicle survival and ovarian steroidogenesis. Silver nanoparticles (AgNPs) are used for their antimicrobial activity in food, home appliances, water treatment and for disinfecting medical devices . Gold naOther studies evaluated the reproductive risks associated with exposure to widely used AgNPs. Cytotoxic responses include increased levels of ROS production, apoptosis, DNA damage and inflammation, although the molecular and cellular mechanisms by which they can exert adverse effects at the organismal level remain elusive . Porcine2NPs and AgNPs  have been detected in the uterus where increased oxidative stress and apoptosis of connective tissues with consequent alteration of contractile activity were observed [The presence of NPs in the uterus can induce developmental toxicity and has deleterious consequences to reproductive performance. In in vivo-exposed animals, TiOobserved ,93,94.2 NPs, administered once every three days from day three of gestation until delivery at 0.02 and 2 mg/Kg, might impair normal fetus development in comparison with higher diameters by causing DNA damage in offspring [It is generally accepted that fetuses are more sensitive to environmental toxins than adults . Recent ffspring . These Nffspring . Most stffspring .It is evident that the indirect toxicity of NPs on placental/fetal functions and long-term health requires further exploration, especially because the key steroidogenic role of placenta during pregnancy could be affected by the suspected endocrine disrupting effects of specific nanomaterials used in NP generation .2 and AuNPs in laboratory animals have increased the appearance of new metastatic sites by increasing the gap between blood vessel cells and allowing cancer cells to migrate more easily to new sites, pointing to the need of accurate studies before using NPS to treat cancers [One of the main problems related to nanomaterials is their ability to induce genotoxic, and thus mutagenic and clastogenic, damage on DNA . The dam cancers .The correct application of cryopreservation technologies is fundamental to improve organ/cell shortage for transplantation. Fertility in young women with cancer but also in healthy young women who electively wish to delay childbearing to avoid the detrimental effects of aging on reproductive capacity. Cryopreservation of germ cells requires the use of specific freezing/thawing cocktails, in which different cryoprotectant agents (CPA) and other chemicals are added depending on freezing procedures (slow freezing or vitrification). Due to its rapid procedure, vitrification is now the most-used method to preserve germ cells and embryos.w/w) of silicon dioxide (SiO2), aluminum oxide (Al2O3), hydroxy apatite (HA) and TiO2NPs (diameter: 20 nm) showed that HA and SiO2 are the most biocompatible nanomaterials having a reduced toxicity. In fact, when used at 0.05%, oocyte survival rate and capacity to complete meiosis up to MII stage were higher in comparison with the other NPs tested and control conditions [3O4NPs  could protect mouse GV oocytes from cryodamage during vitrification procedures. After IVM and IVF, the quality of frozen/thawed embryos has been found to be positively modulated by presence of these NPs [Unfortunately, the survival rate of vitrified oocytes is still low (about 4%) , but recnditions . Similarnditions . More rehese NPs .Another interesting aspect of female fertility preservation in which NPs are starting to use is contrasting ovarian aging. Despite the existence of various strategies, no clinical treatments are currently available to delay ovarian aging. Drug delivery via NPs should be effective in postponing ovarian aging. In fact, studies are currently being pursued to use them in both 3D cultures and tissue engineering, with the purpose of increasing cell proliferation and viability .A significant improvement in the safety of NP-delivered drugs in cancer patients has been achieved in recent years to bypass the limitations of chemotherapy. NPs are utilizable for biomedical applications to facilitate the absorption of drugs that can be bound on their surface ,111. To Recently, the application of RNAi-based therapies has been sharply accelerated because a significant decrease in nonspecific toxicity can be obtained by encapsulating multiple payloads of drugs within nanocarriers and by monitoring si-RNA delivery by noninvasive imaging techniques. Nano-siRNA drugs can be of great help in overcoming the limitations of chemotherapy. Recently, NP-mediated siRNA delivery strategies such as polymeric- and lipid-based systems, rigid nanoparticles and NP coupled with specific ligand systems have been tested. NP-based codelivery of anticancer drugs and siRNA targeting various mechanisms of multi-drug resistance is a cutting-edge strategy for ovarian cancer therapy. Since siRNAs that are negatively charged are unable to cross cell membranes, the use of nanocarriers allows for their increased cellular uptake . One exaThe fact that NPs can harm biological systems but can be used for innovative therapies capable of conferring health benefits highlights the two sides of a coin . To less


Two of the molecular families closely associated with mediating communication between the brain and immune system are cytokines and the kynurenine metabolites of tryptophan. Both groups regulate neuron and glial activity in the central nervous system (CNS) and leukocyte function in the immune system, although neither group alone completely explains neuroimmune function, disease occurrence or severity. This essay suggests that the two families perform complementary functions generating an integrated network. The kynurenine pathway determines overall neuronal excitability and plasticity by modulating glutamate receptors and GPR35 activity across the CNS, and regulates general features of immune cell status, surveillance and tolerance which often involves the Aryl Hydrocarbon Receptor (AHR). Equally, cytokines and chemokines define and regulate specific populations of neurons, glia or immune system leukocytes, generating more specific responses within restricted CNS regions or leukocyte populations. In addition, as there is a much larger variety of these compounds, their homing properties enable the superimposition of dynamic variations of cell activity upon local, spatially limited, cell populations. This would in principle allow the targeting of potential treatments to restricted regions of the CNS. The proposed synergistic interface of ‘tonic’ kynurenine pathway affecting baseline activity and the superimposed ‘phasic’ cytokine system would constitute an integrated network explaining some features of neuroimmune communication. The concept would broaden the scope for the development of new treatments for disorders involving both the CNS and immune systems, with safer and more effective agents targeted to specific CNS regions. Many neuropsychiatric disorders are accompanied by abnormal regulation of the immune system, while many immune system disorders are influenced by the central nervous system (CNS) . The bi-Despite this overlap, neither the immune system mediators nor tryptophan metabolites alone provide a complete explanation for disorders which may involve both systems. It is therefore proposed that the two families of compounds function as an integrated inter-dependent network in which the broad changes in neural excitability and synaptic plasticity in the CNS, and the generalized control of immune system inflammatory balance and tolerance are modulated by kynurenines. This activity would determine the basal level of cell function across the neuroimmune divide. Cytokines and chemokines could then superimpose a layer of greater regional and functional refinement which generates the specificity required for organ and tissue selective bi-directional communication.This perspective begins with a brief summary of kynurenine and cytokine biology, followed by a more detailed expansion of the integration concept with examples of interactions between cytokines, kynurenines, the CNS and immune system. The concept of ‘volume transmission’ in the CNS is discussed as the structural basis within which those molecular interactions can occur, with comments on the contribution of the blood-brain barrier (BBB) and glia. Finally, there is an exploration of the possible mechanisms by which the different actions of tryptophan metabolites and immune mediators can achieve the functional inter-dependence and tissue specificity for a neuroimmune interface with regional selectivity.IL1B  with symptoms of hyperthermia, apathy, reduced locomotion and socialization, and a centrally mediated loss of appetite, all indicating CNS involvement . InterleIL1B . AntagonIL1B .Other cytokines can affect the CNS , some exChemokines (“chemotactic cytokines”) govern the movement and localization of cells  and are L-kynurenine primarily by indoleamine-2, 3-dioxygenase-1 (IDO1) (The kynurenine pathway  accounts1 (IDO1) . Interfe1 (IDO1) . This wa1 (IDO1)  and the 1 (IDO1) , 2002, pActivation of the kynurenine pathway can be induced by molecular products of tissue injury or microbial fragments such as lipopolysaccharides (LPS), often acting via TLRs . These “N-methyl-D-aspartate (NMDA)  . The fune (NMDA) . When ove (NMDA) .antagonist at all three glutamate receptor subtypes responding respectively to NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) or kainic acid   or the GThe kynurenine pathway is present constitutively in APCs such as macrophages and dendritic cells . The intAlthough the first enzyme of the kynurenine pathway – IDO1 – is induced primarily by IFN-γ , activatCytokines associated with anti-inflammatory resolution and tissue recovery, such as IL-4 and IL-10 inhibit IDO1 expression , while IIFN-γ also induces KMO, KAT and kynureninase in circulating monocytes  and DCs,Exposure to stress induces expression of cytokines and IDO1 . Mice laIDO2 is less active than IDO1 but mediates the same metabolism of tryptophan to kynurenine  and may Tryptophan is also metabolized to kynurenine by tryptophan-2,3-dioxygenase (TDO2) an enzyme with greater substrate selectivity for tryptophan. TDO is expressed mainly in the liver , 2018, bArguments about the roles of tryptophan catabolites and cytokines in the CNS often center around the relationship between their systemic and central concentrations. Several studies of blood and CSF cytokine concentrations have shown parallel qualitative and quantitative changes, including those on TGF-β  with oneHowever, levels of cytokines in the tissues and organs can be highly variable. A cytokine panel used in patients with tuberculous meningitis indicated that all (except IL-10) were present in brain at levels lower than in CSF , while aIn the case of kynurenines, many studies of depression have reported increased activity along the kynurenine pathway but this is not a universal observation . There iOverall, therefore, the data suggest that the CNS presence of complex molecules does not lend itself to meaningful correlations with symptoms or with the levels of other compounds – such as kynurenines – with very different permeabilities and kinetics. These difficulties may disappear under the present hypothesis, as discussed in depth below, since the essential correlations to examine are not between cytokines or between kynurenine metabolites, but are the cross-correlations between the two families of compounds, reflecting the complementary nature of their contributions. Such measurements of course still need the appropriate attention to diagnosis, cohort variations, and clearer definition of the CNS regions relevant to a particular disorder or symptom.Clearly, the cytokine and kynurenine families play fundamental and wide-ranging roles in the immune system and CNS, with confusion arising on how to relate either of these groups to disease symptoms or severity. The hypothesis proposed here is that the two sets of compounds exert complementary actions so that integration between them is required to explain aspects of disease. This idea will be expanded in the following sections by considering in more detail how that integration could arise, with an emphasis firstly on molecular interactions and then secondly on spatial factors. Subsequent sections will then discuss the structural and functional aspects of how and where these interactions can occur, focusing on volume transmission and the roles of glial cells and the BBB.Some of the more prominent interactions between cytokines or chemokines and the kynurenine metabolites of tryptophan are summarized in Immune mediators can interact with glutamate-mediated transmission. TNF, for example, enhances release of glutamate from neurons and enhances its exocytotic release from glia . Some ofStimulation of NMDAR promotes TNF expression  and TNF Interactions with AMPAR are less clear, since TNF modulates the expression and activity of AMPAR  and TNFRAMPAR expression . However+ T cells, promoting differentiation to regulatory T cells (Tregs), but suppresses Retinoic Acid Receptor-related Orphan Receptor-γt (RORγt) which promotes differentiation to Th17 inflammatory cells  and leading to reduced proliferation and increased apoptosis . ActivatThe effects of kynurenine are cell and context-dependent since it enhances IL-6 and IL-13 production by mast cells but inhibits IL-13 release from iNKT cells probably via AHR . This maAn excellent example of the intimate functional relationship between immune mediators and kynurenine metabolites is the sickness behavior, described above. While early work implicated IL-1β, the behavior is also prevented by IDO1 inactivation  emphasiz+T cells  . 3HAA enNK cells  while prNK cells .+ CD8+ cells.The ratio between anthranilic acid and 3HAA correlates with inflammatory status in many disorders  and 3HAAInflammatory activation of IDO in peripheral cells will increase plasma levels of kynurenine and 3HK which enter the CNS by diffusion and by the amino acid transporter LAT-1 (SLC7A5) . KynurenImportantly, quinolinic acid induces the expression of several key chemokines  and chemokine receptors  . The res+ cell differentiation to the Th17 phenotype via AHR  but some regional specificity is required to produce the varied, dynamic regulation of different behaviors, sensations (including pain), emotional reactions, and intellectual function, resulting from activity across the CNS and immune system. While most actions of kynurenine and its metabolites will be a generalized regulation of CNS development, excitability and plasticity, together with an organism-wide modulation of immune function, the present hypothesis argues that the reciprocal effects of immune system mediators on the CNS will produce a more localized, focused control of neuronal and glial activity. A mechanism is required to explain how such anatomical and functional complementarity could be generated between two such apparently complex and diffuse environments as the immune system and CNS.Certainly one factor would be the distribution of receptors. Kynurenine pathway targets are widespread, including the glutamate receptors expressed by CNS neurons and glia, and many populations of leukocytes can be activated by quinolinic acid and blocked by kynurenic acid. Alternative targets for kynurenate such as AHR and GPR35 are present in a large fraction of cells, while effects on oxidative processes and redox status will affect almost all cell types. A highly specific distribution of receptors was first noted for IL-1  but has Viewed together, these various considerations contribute to the ability of individual cytokines to define specific aspects of CNS or immune system function superimposed on the more generalized regulation of cell behavior produced by the kynurenine pathway. This view could form the basis of selectivity mechanisms already recognized in peripheral tissues but now applied to the CNS, such as homing.The term ‘homing’ refers to the directed migration of different cell types to appropriate locations affected by environmental or pathological changes. The concept is well established in peripheral tissues with the differential targeting of cells to various tumors or specific cell types within tumors. The homing factors are primarily cytokines and chemokines . The rol+ T cells to the intestinal mucosa, and inhibiting its formation suppresses the phenomenon. The homing of CD4+ T cells is partly regulated by the interaction between the two T cell differentiating transcription factors FoxP3 and RORγt. The former complexes with AHR to enhance homing while RORγt inhibits homing .Contact complementarity may resemble the spatial location of cells determined by their specific mix of proteins. In mouse brain, for example, microglia have high levels of CD11b, F4/80 and FcγRI in the hippocampus whereas in the frontal cortex CD11b is lower and CX3CR1  is higher . The difThe ability of the proposed mechanism to account for neuroimmune selectivity can be illustrated mathematically. If a group of leukocytes interacted with CNS cells based on the expression of a single molecule then, based on the existence of 100 relevant cytokines and related molecules influencing the CNS, there could be around 100 neuro-immunologically distinguishable regions of CNS. However, since the cell interactions could involve any number of mediators, in different combinations and at densities which may vary in time, there could be many millions of distinguishable CNS regions. This variety could generate localized activity affecting small groups of CNS neurons depending on the state of the cells concerned, local physiological inputs and the specific local extracellular chemical composition. It is this spectrum of contacts which could be prominently affected by the widespread changes in cell activity and polarization produced by environmentally modulated changes in IDO1 expression and its tryptophan metabolites.Not surprisingly there will be exceptions to these rules, as some cytokines show a degree of non-selectivity for their receptors. Although some pairings are highly specific, as with the interaction between CXCL12 and its receptor CXCR4, some ligands can affect several receptor populations, and individual receptor subtypes may respond to multiple ligands . This imEven the relatively few leukocytes within the CNS parenchyma or meninges would contribute to this regional complexity, since their products would access receptors on thousands of CNS cells (around 1 million per microliter). Complexity would be further enhanced by the diffusion and dilution gradients of compounds from several leukocytes.In the CNS, the expression of complementarity factors will be influenced by the level of local neuronal activity . SimilarThe overall outcome of these interactions will be to generate a complex intercellular chemical matrix which would generate a different ‘map grid reference’ for each point within the CNS, rather like the point-to-point representations of the body surface in the sensory neocortex. The degree of commonality or overlap between cells in this chemical map will then (as for a zip or post code) determine the size and composition of each region.There may also be a temporal aspect to cytokine-kynurenine interaction. Activation of IDO1 can occur over the first few hours of an appropriate stimulus, remaining elevated for 24 h or more, whereas IL-1β, IL-6 and TNF decline much more rapidly . EnzymesIn contrast, cytokines can exhibit a very wide range of time courses which are likely to amplify functional differences between the cytokines, as for IL-1 or IL-6 , IL-18 , IL-4 anThe existence of such a breadth in the temporal range of cytokine expression and activity would overlap that of the kynurenine pathway, such that the initiation, development or termination of cytokine activity could be modulated by the more generalized effects of kynurenine and its metabolites. This gives rise to the proposal that kynurenines provide a continually shifting ‘tonic’ baseline of cell activity, onto which the ‘phasic’ effects of immune system mediators are superimposed.If, as proposed here, the kynurenine pathway and cytokines function in a complementary fashion, partly accounting for the weak or conflicting correlations between tissue levels of either family of compounds in relation to disease symptoms or severity, how and where does this integration occur? This section addresses the question by considering the most important concept of ‘volume transmission,’ with additional comments on the contributions of glial cells and the BBB. Together, these features define the physical, structural factors involved in the neuroimmune interface.The classical concept of synaptic transmission by fast neurotransmitters was that they acted at tightly apposed synaptic junctions, on a timescale of milliseconds, to modulate the polarization of neurons. Using the dominant amino acid glutamate as an example, fast transmission was mediated by AMPA and kainate receptors, with a slower excitation by NMDARs which generated much of the plasticity-based changes underlying behavior and cognition . The subThis view became modified by the recognition that slow neurotransmitters such as the monoamines were often released from axonal varicosities into a much larger volume of extracellular space, and this idea evolved further into the concept of “volume transmission” . The terSince CNS cells secrete and respond to a variety of growth factors and cytokines, including IL-1β, IL-6, and TNF in addition to conventional neurotransmitters and neuromodulators , volume transmission will generate a highly complex medium in which active compounds can modulate the activity of neurons and glia with the appropriate receptors or targets at much greater distances than across synapses . The celIndeed, a central tenet of the neuroimmune concept is that leukocyte populations will participate in these chemical exchanges, responding to neuroglial secreted transmitters, modulators and cytokines, but also adding their various products of cytokines and proteins into the extracellular matrix. This will apply to resident and itinerant leukocytes in the CNS, so that the information on neuron and glial activity represented by the chemical composition of the CNS extracellular medium will be imprinted onto the cohort of leukocytes present in the CNS at that time. As those leukocytes eventually return to the systemic circulation, any subtle changes in phenotype will influence their interactions with other immune system cells, modifying their activity or differentiation and proliferation such that even a small number of cells ‘conditioned’ by the CNS extracellular medium could have a significant impact on immune system function.Equally, of course, the molecular information in leukocytes entering the CNS will be determined by the state of the immune system in the previous minutes or hours, allowing the imprinting of that information onto neuron and glial activity.It has been suggested that the level of local neural activity influences cytokine production and receptor expression which then generate a differential attraction of immune system cells into the CNS, further linking neural activity with neuroimmune communication . Such a In addition to the release and uptake of molecules by transporters or diffusion, cells may secrete high molecular weight compounds such as proteins and nucleic acids packaged into vesicles (exosomes) up to 100 nm diameter . TransfeIn addition to the complexity of volume transmission, the presence of a BBB will itself modify the movement of compounds to and from the CNS , contribAs with much larger proteins, cytokines (including chemokines) can also enter the CNS through leaky regions such as the circumventricular organs (CVO) . Some, iSome smaller compounds ‘leak’ across the capillary endothelial layer outside the CVO to reach biologically active concentrations within the CNS. A classical example is morphine which crosses the barrier poorly but still sufficiently to induce a rapid and effective analgesia . Some coIntact leukocytes can cross or by-pass the BBB giving them, and the cytokines they express, direct access to the CNS parenchyma. In particular, elements of later stage, adaptive immune activity including T cells are able to access the CNS via diapedesis across the choroid plexus and by the binding between chemokine CCL20 and its chemoattractant receptor CCR6 . This sy+ T-cells, with evidence that marked changes of learning are associated with neuronal loss in the hippocampus following the depletion of peripheral CD4+ T cells, implying that the presence of these cells makes a significant contribution to neuronal and synaptic plasticity  which can occur physically associated with the GluN2B subunit of NMDARs . Both ILA novel perspective on the neuroimmune relationship has been gained from interfering with the D3 dopamine receptor, which is localized exclusively to CNS microglia. Blockade or deletion of D3 caused activation of those glia, with symptoms of depression and increased levels of IL-1β, IL-6, and TNF in dopaminergic neurons .The discussion of immune system effects on the CNS is probably easier to conceptualize than the converse interaction, so it is important to note experimental examples showing how CNS activity directly influences the immune system.The reciprocal production of secreted neuro-glial transmitters , neuromoIntracerebral injections of NMDA increased the number of NK cells in the systemic circulation, with increased levels of TNF. These responses were reduced by mild stress acting on CNS efferent glutamate-mediated (NMDAR-activated) pathways demonstrating their ability to influence systemic immunological function .Mild stress also increased CNS microglial activation and cytokine expression . However+T cells is involved in the dopaminergic regulation of multiple sclerosis, arthritis, inflammatory bowel disease and Parkinson’s disease  modulates cytokine release and IDO expression in murine and human cells is an important extension to this concept. It reveals that a microbially important molecule, derived from the metabolism of tryptophan and which is not produced by mammals, can have highly significant effects on the host immune system , even though counseling can be more effective . HoweverThe concept of a neuroimmune interface attempts to explain how systemic insults can affect brain function and how aspects of behavior affect the immune system. The kynurenine pathway is viewed as pivotal in these interactions, mediating the translation of CNS neural activity to changes in the surveillance, defensive and tolerance activities of the immune system. Conversely, the latent presence of the kynurenine pathway in cells of the immune system, under the control of cytokines and chemokines, enables immune system activity to influence the CNS. Similarly, activity in peripheral immune cells, with the immunologically active and neuroactive factors they produce represents an additional force modulating neuronal and glial functions, contributing to subtle changes in a variety of behaviors. Overall, the low intensity modulation of neuronal excitability and synaptic plasticity by glutamate, NMDARs and the kynurenine pathway, it is proposed, lays the shifting foundation of a ‘tonic’ modulation of neuronal and glial activity upon which the ‘phasic’ effects of immune system modulators are superimposed at a localized, perhaps even cellular, level. Efforts to explain clinical disorders and to develop new diagnostic tools or treatments should take into account both the immune system mediators and kynurenine pathway metabolites for a more complete explanation and understanding of the neuroimmune and immuno-neural interface. The potential for producing highly localized changes in selected CNS regions could improve the safety, tolerability and efficacy of drugs for a range of psychiatric and neurological disorders as well as autoimmune conditions.TS wrote the initial draft. FC, Y-SH, N-YC, LD, RW, and TS contributed to the subsequent discussions and modifications. All authors contributed to the article and approved the submitted version.


Human leukocyte antigen (HLA) genes have been implicated in cancer risk and shared heritability of different types of cancer. In this immunogenetic epidemiological study we first computed a Cancer-HLA profile for 30 cancer types characterized by the correlation between the prevalence of each cancer and the population frequency of 127 HLA alleles, and then used multidimensional scaling to evaluate the possible clustering of those Cancer-HLA associations. The results indicated the presence of three clusters, broadly reflecting digestive-skin-cervical cancers, reproductive and endocrine systems cancers, and brain and androgen-associated cancers. The clustering of cancer types documented here is discussed in terms of mechanisms underlying shared Cancer-HLA associations. It is now well-established that genetic factors are not only a significant contributor of cancer risk but also contribute to shared heritability among different cancers8. For instance, one study evaluating the genetic correlation between six cancer types in individuals of European ancestry found significant genetic correlations of colorectal cancer with pancreatic cancer, breast cancer, and lung cancer as well as genetic correlations between lung cancer and breast cancer6; in contrast, they found no evidence of shared heritability between prostate cancer and the other cancers they investigated. A more recent study from the same group reported statistically significant genetic correlations between breast cancer with ovarian cancer, lung cancer, and colorectal cancer as well as lung cancer with head/neck cancer7. A study evaluating 13 cancer types found the strongest genetic correlations between kidney and testicular cancer, diffuse large B-cell lymphoma and osteosarcoma and chronic lymphocytic leukemia, and bladder and lung cancer5. Finally, a study of 18 cancer types in two large cohorts of European ancestry found several genetically associated cancer pairs including positive correlations between colon and rectal cancers; esophageal/stomach cancer and Non-Hodgkin’s lymphoma, breast, lung, and rectal cancers; bladder and breast cancers; melanoma and testicular cancer; and prostate and thyroid cancers8. In addition they reported four negative correlations including endometrial and testicular cancers; esophageal/stomach cancer and melanoma; lung cancer and melanoma; and Non-Hodgkin’s lymphoma and prostate cancer8. Moreover, there was evidence of widespread pleiotropy including 25 regions that were associated with more than 1 cancer type, 14 of which involved the Human Leukocyte Antigen (HLA) region8.Cancer, a leading cause of death worldwide13. Moreover, HLA has recently been implicated in the shared heritability across cancers with some loci evidencing unidirectional associations with cancer types and others demonstrating discordant associations with cancer types, highlighting varying associations between HLA and different types of cancer8.The HLA region of chromosome 6 codes for cell-surface proteins involved in immunosurveillance and T-cell activation aimed at elimination of tumor cells and pathogens. Specifically, Class I HLA molecules  present intracellular antigen peptides to CD8+ cytotoxic T cells to signal destruction of infected cells whereas HLA Class II molecules  present endocytosed extracellular antigen peptides to CD4+ T cells to promote B-cell mediated antibody production and adaptive immunity. HLA has been increasingly implicated in various types of cancerTaken together, prior studies have identified shared genetic associations between some cancer types and implicate HLA as common genetic mechanism. HLA genes are the most highly polymorphic of the human genome. Here we take advantage of the population heterogeneity of HLA and extend prior lines of research by evaluating similarities between population-level Cancer-HLA associations involving 30 types of cancer and 127 HLA alleles. This approach captures the complex associations between HLA and cancer and permits clustering of cancers with similar HLA profiles, potentially permitting identification of common genetic mechanisms underlying clusters of cancers.14, a publicly available catalog of data from the Global Burden of Disease study, the most comprehensive worldwide epidemiological study of more than 350 diseases. The number of people with each cancer in each country was divided by the total population of each country in 201615 and expressed as a percentage. We have previously shown that life expectancy for these countries are virtually identical16; therefore, life expectancy was not included in the current analyses.The population prevalence of the 30 cancers Table  in 2016 18 on October 20, 2020. As we reported previously16, there was a total of 2746 entries of alleles from the 14 CWE countries, comprising 844 distinct alleles, i.e. alleles that occurred in at least one country. Of those, 127 alleles occurred in 9 or more countries and were used in further analyses. This criterion is somewhat arbitrary but reasonable; it was partially validated in a previous study19.The frequencies of all reported HLA alleles of classical genes of Class I  and Class II  for each of the 14 CWE countries were retrieved from the website allelefrequencies.net The distribution of those alleles Table  to the HStatistical analyses were performed using the IBM−SPSS package  and Intel FORTRAN .HLA profiles for each cancer were derived by computing, first, the Pearson correlation coefficient, Negative P/S estimates indicate a protective association , whereas positive P/S estimates indicate a susceptibility association . Thus 30 Cancer-HLA profiles were computed, each consisting of 127 values of S-stress = 0.001, minimum s-stress value = 0.005, maximum iterations = 30.A MDS analysis of the Cancer-HLA matrix was performed using the ALSCAL procedure of the IBM-SPSS statistical package (version 27). More specifically, we used (a) the individual differences (weighted) Euclidean distance model, where each HLA allele contributed separately to the solution, (b) Euclidean distance as the distance measure, (c) ordinal level of measurement, (d) solution in 2 dimensions, and (e) the default criteria for convergence: 20, as follows. Given N = 30 points (cancer types), we computed 30 distances to nearest neighbor, The MDS analysis yielded a 4 × 4  2-dimensional cancer configuration map, where each cancer type occupied a point in the map based on its X–Y (Dimension 1 and 2) coordinates. The initial step in the spatial analysis of the distribution of cancer types in this map was to test the null hypothesis that the spatial distribution of the 30 cancer types was random. For that purpose, we used the distance to nearest neighbor measure of Clark and EvansNext, we computed the density, The mean distance to nearest neighbor expected in an infinitely large random distribution of density With a standard error ofThe ratio We performed a K-means clustering analysis of the location of cancer types in the MDS map in order to identify possible clusters using the iterate-and-classify method of the K-means Cluster Analysis procedure of the IBM-SPSS statistical package (version 27).This article does not contain any studies with human participants performed by any of the authors.The MDS cancer type configuration map is shown in Fig. The following values were obtained:The high value of normal deviate nt}c Eq. , and thementclass2pt{minim21). Therefore, we performed a K-means clustering analysis which assigned the 30 cancer types to 3 clusters , attesting to the congruence of existing data from other studies. Finally, the immunogenetic association between mesothelioma and melanoma P/S is illustrated in Fig. The results above are based on associations between immunogenetic HLA-Cancer profiles of specific cancer types. It would be important to cross-validate this approach by comparing associations above with known associations between cancer types occurring in the same individual. To that end, we used 4 pairs of cancers with significant probabilities of co-occurrence in the same individuals and computed the correlations between their immunogenetic HLA-Cancer profiles to test the prediction that those correlations would be positive, high, and statistically significant, if, indeed, the immunogenetic predictions are congruent with the documented co-occurrence. The 4 pairs of cancers included (1) multiple myeloma and kidney cancer 28 Fig. , (3) pro 30 Fig.  for whicair Fig.  for whic36 to identify clusters of cancers based on the population-based HLA profile of each cancer in 14 countries in Continental Western Europe. The findings, which indicate the presence of three clusters derived from the immunogenetic HLA-cancer associations, partially overlap with prior studies documenting shared genetic associations for different cancers8 and provide novel insights regarding potential shared mechanisms underlying different types of cancers. Remarkably, the immunogenetic associations derived here were congruent with known associations between the co-occurrence (or the significant probability of co-occurrence) of certain types of cancers, thus lending credit to our immunogenetic approach.Here we used multidimensional scaling37, epitopes common to different antigens, and/or viral oncoproteins39 associated with several types of cancer within a cluster. Additional research is warranted to identify the specific factors linking cancers within each cluster and those that distinguish different clusters.The three clusters derived Fig.  are base7, breast cancer and ovarian cancer7, kidney and testicular cancers5, and esophageal cancer and Non-Hodgkin’s lymphoma8. The present findings provide additional support regarding shared genetic associations between these cancer types and specifically point to highly similar HLA as partially driving those associations. Prior studies evaluating genetic associations between different types of cancer have been limited by inclusion of relatively few types of cancer and very few studies have specifically evaluated HLA with regard to genetic associations. Here we included 30 different cancers and 127 different HLA alleles permitting identification of overlapping cancer-HLA associations that have not previously been reported to our knowledge. For example, we found that prostate cancer and melanoma are very tightly clustered, implicating HLA in that shared risk. Prior studies have documented a possible link between melanoma risk and prostate cancer risk, purportedly associated with androgen-dependence41. Esophageal cancer and non-Hodgkin’s lymphoma, which were clustered with melanoma and prostate cancer here, have also been linked to androgens43. Though intriguing, a mechanism linking HLA to androgen-associated cancers remains to be elucidated.Several associations documented here overlap with those of previous studies and several others are novel. Consistent with the clustering observed in our study, previous studies have documented genetic associations between several types of cancer including breast cancer and lung cancer45. For instance, the genetic architecture of lung cancer has been shown to vary by population and environmental exposures5. Furthermore, although environmental factors play a role in risk of cancers3, we solely focused on the influence of HLA and did not evaluate the influence of environmental exposures in the present study. Nonetheless, the results provide compelling evidence regarding the influence of HLA on the population clustering of different types of cancer and suggest that additional research evaluating Cancer-HLA associations is warranted. As discussed above, HLA relates to antigens, and, as such, HLA-based measures and associations are necessarily discussed in the context of antigen presentation and processing by HLA. Given the diversity of proteins produced by tumor cells, the concomitant degradation of host proteins, and the added potential viral proteins implicated in several cancers, it is not surprising that the HLA-Cancer immunogenetic profiles provide a broad spectrum of cancer protection/susceptibility estimates reflecting the footprint, so to speak, of a specific cancer with regard to the antigens related to the cancer. In that sense, the observed associations and clustering described here furnish a framework within which a 127-dimensional HLA allele map is effectively reduced to a 2-dimensional map by multidimensional scaling that allows for a simplified investigation of immune-related associations among cancer types. Since the origin of the various antigens involved can be very diverse, as mentioned above, the associations found among the various cancer types are more holistic in nature, and their interpretation rests on information stemming from considerations regarding the antigens themselves. For example, a major part of the co-occurrence of pancreatic and ovarian cancers stems from the presence of BRCA genes in individuals susceptible to both cancers but their corresponding significant immunogenetic association indicates that the spectrum of antigens related to those cancers is very similar in those cancers despite the fact that they affect very different cells (pancreas vs. ovaries). Similar considerations hold for the more tenuous attribution of the mesothelioma/esophageal cancer association to asbestos and of the gallbladder/colorectal cancer association to gallstones. A recent study linking mesothelioma to melanoma46 via BAP1 gene highlights the predictive power of our immunogenetic approach which showed a high correlation between mesothelioma and melanoma HLA P/S estimates  but which produce a spectrum of antigens that engage in a similar way the HLA system. Given that the success of immune blockade immunotherapy for cancer partly depends on the HLA genetic makeup of the patient