Background: Over the past decades, advancements in oncological treatments have led to major improvements in survival. Particularly for adolescents and young adults (AYAs), fertility is an important concern in cancer survivorship. The purpose of the review is to provide physicians with a practical overview of the current knowledge about the impact of systemic oncological treatments on the fertility of female and male AYAs. Methods: A systematic review was performed based on relevant articles obtained from 4 databases up until 31 December 2022. Results: The mechanisms of gonadotoxicity and the concurrent risk is described for the following categories: chemotherapy, targeted therapy and immunotherapy. For the category “chemotherapy”, the specific effects and risks are listed for the different classes and individual chemotherapeutics. In the category “targeted therapy”, a distinction was made between tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Information concerning immunotherapy is scarce. Conclusions: The effects of chemotherapy on fertility are well investigated, but even in this category, results can be conflicting. Insufficient data are available on the fertility effects of targeted therapy and immunotherapy to draw definitive conclusions. More research is needed for these therapies and their evolving role in treating cancers in AYAs. It would be useful to include fertility endpoints in clinical trials that evaluate new and existing oncological treatments. Cancer in adolescents and young adults (AYAs) is defined by the National Cancer Institute and by the working group of the European Society for Medical Oncology (ESMO) and the European Society for Paediatric Oncology (SIOPE) as diagnoses occurring among those aged between 15 and 39 years . In 2019As treatments for malignancies become increasingly successful and survival rates for many types of cancer improve, although, at a slower pace in AYAs, more and more cancer patients become cancer survivors . SpecifiThe field of systemic cancer therapy is continuously evolving, and next to classic cytotoxic chemotherapy, targeted therapy and immunotherapy have become increasingly incorporated into the standard of care. The impact of chemotherapeutic agents on fertility is best described in the literature, comprising varying degrees of gonadotoxicity depending on the mechanism of action, dose and length of treatment, and age of the patient at the time of treatment. For other systemic cancer treatments, the harmful effects on fertility are less known ,8,9.Due to gonadotoxic treatment, primary ovarian insufficiency (POI) can occur as a long-term effect of anti-cancer treatment. POI is defined as the loss of normal ovarian function before a woman reaches the age of 40. It is characterized by secondary amenorrhea due to hypergonadotropic hypogonadism. Other symptoms of POI are caused by estrogen deficiency, for example, hot flashes, sweating at night, mood swings/disorders, sleep disorders, lack of concentration, arthralgia and reduced sexual function. It is also associated with loss of bone mineral density and a higher risk of cardiovascular events .Patients should be well-informed about gonadal function and fertility before and after treatment and offered different strategies for fertility and fertility preservation ,8,11,12.This systemic review is based on a literature search in 4 databases, including PubMed, Embase, Google Scholar and Web of Science. Articles published in English until 31 December 2022 were considered. This manuscript also includes articles from decades ago because of their lasting relevance up to now. The population of interest consists of adolescents and young adults, defined in this review as ‘people between 15 and 39 years old at cancer diagnosis’. Therefore, all articles about patients of this age were included. Articles in which some patients did not fit these specific age limitations, but others did not, were also included. When the age was not specifically mentioned, but it could be assumed from the wording that the article was about AYAs, these articles were also included. When selecting specific types of treatment and treatment schedules, special attention was given to treatments for cancer types specific to the above-mentioned population. Regarding immunotherapy, this review focuses on immune checkpoint inhibitors because these are widely used in current clinical practice compared with other treatments, such as CAR-T cell therapy, tumour-infiltrating lymphocytes or cancer vaccines. Animal studies were excluded. Preclinical studies were only considered when focussing on human patients. Review articles were only included if they contained extra relevant information compared with the original articles.The search terms were ‘chemotherapy/targeted therapy/immunotherapy/monoclonal antibodies/tyrosine kinase inhibitors’ and/or ‘cancer’ and/or ‘cancer treatment’ and/or ‘(onco)fertility’ and/or ‘guidelines’ and/or ‘reproductive health’ and/or ‘gonadotoxic effects’ and/or ‘gonadal function ’ and/or ‘semen analysis’ and/or ‘fertility damage’ and/or ‘side effects’ and/or ‘gonadotoxic impact’ and/or ‘adolescents and young adults/AYA’. Articles that fit the aforementioned criteria were considered for inclusion. If an article did not provide the right focus, it was excluded. The article search is illustrated in the PRISMA flowchart . The revThis review is registered in the PROSPERO registry under number 409256.At birth, the ovaries have about 1 to 2 million oocytes forming the ovarian reserve, of which only 300 will reach maturity during reproductive life. The oocyte count progressively decreases until menopause ,14. The The ovarian reserve determines a woman’s fertility potential, both for spontaneous conception and pregnancy, through assisted reproduction techniques. Several markers are used to estimate ovarian reserve, such as anti-Müllerian hormone (AMH) levels ,16 and aIn general, gonadotoxicity is expressed as the risk of developing permanent amenorrhea. It is divided into three classes: high risk, intermediate risk and low to very low risk, with a chance of permanent amenorrhea of more than 80%, 20–80% and less than 20%, respectively . The impact of chemotherapy on fertility has been known and reported since the 1970s ,21. ChemChemotherapeutic agents act through different mechanisms in the ovary . Some acThe short-term effects are responsible for the loss of growing follicles and cause amenorrhea soon after the beginning of chemotherapy ,23,25,26The long-term effects are responsible for the loss of ovarian reserve and subsequent POI and infertility . ChemothFirst, the loss of primordial follicles can be caused by direct DNA alterations or by accelerated recruitment. For alkylating agents and anthracyclines, DNA alterations and double-stranded breaks can lead to apoptosis of primordial follicles. Although, some follicles will be able to repair DNA damage and survive. In the case of platinum-based compounds, there seems to be no direct toxicity in primordial follicles . AnotherSecond, vascular injury, fibrosis and stromal damage through chemotherapy-induced ischemia and inflammation can indirectly affect ovarian endocrine function ,29. OvarOver the past few decades, personalized medicine, based on the underlying molecular and genetic characteristics of the tumour, has made its way into cancer treatment. These targeted therapies have not only improved the overall survival of oncology patients but also generally have less toxicity compared with conventional chemotherapy . The litMonoclonal antibodiesMonoclonal antibodies against human epidermal growth factor receptor 2 , e.g., trastuzumab and pertuzumab, are widely used in HER2-positive breast cancer and more recently also in other HER2-positive cancer types such as gastro-intestinal tumours ,39,40. TTyrosine kinase inhibitorsTyrosine kinase inhibitors (TKIs) are a class of small molecule targeted therapy inhibiting cell signalling by blocking cell surface tyrosine kinase receptor phosphorylation. As many of these tyrosine kinases and receptors are involved in regulating oogenesis, primordial follicle activation, folliculogenesis and corpus luteum formation, inhibiting them may hamper normal ovarian function. The best-described tyrosine kinases and receptors that play a role in ovarian function are tyrosine-protein kinase (c-KIT), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) ,37.Targeting the c-KIT receptor, expressed on the ovarian surface, leads to the inhibition of cell proliferation and apoptosis. PDGFR-a is found on primordial follicles, and TKIs that block the PDGFR-a receptor disrupt follicular development resulting in a decrease in total follicle count. These effects are reversible, and there are reports of women conceiving while on TKI, indicating that at least some gonadal function is preserved . AlthougAnother potential mechanism of indirect interference with normal ovarian function is the possible effect of TKIs on endocrine function, in particular, thyroid dysregulation, as this is associated with adverse fertility outcomes ,43. InhiBussies et al. add, however, that kinase inhibitors that target mTOR  or the BCR-ABL (breakpoint cluster region-abelson) fusion protein seem to be protecting the ovarian reserve in patients undergoing chemotherapy. Inhibition of mTOR appears to temporarily halt ovarian cell activation making them less vulnerable to the toxic effect of chemotherapy .The effects of TKIs on ovarian function seem to be reversible after treatment interruption, but an optimal wash-out period is unclear . InteresOtherPoly ADP-ribose polymerase (PARP) inhibitors are used in BRCA-mutated cancers and interfere with DNA damage repair as PARP enzymes repair single-strand DNA breaks. It is possible that PARP inhibition causes loss of primordial follicles due to loss of DNA repair and therefore impairs fertility .Proteasome inhibitors, matrix metalloproteinases, heat shock protein inhibitors, and promotors of apoptosis are relatively new classes of small molecule inhibitors. The potential effects of these agents on the female reproductive system are not described .The discovery of immunotherapy with immune checkpoint inhibitors (ICI) has improved the survival of oncology patients, both in metastatic and adjuvant settings . ICIs arInherent to treatment with ICI are immune-related adverse events (irAEs), which can affect basically any organ, including the endocrine system . EndocriThe following sections describe the gonadotoxic effects of each class of chemotherapy. Alkylating agentsAlkylating agents  significantly contribute to POI and infertility and are considered to be the most gonadotoxic chemotherapeutics . Their t2 did not seem to be at significantly increased risk of fertility impairment. Results of this analysis in female childhood and AYA cancer survivors showed that cumulative doses of the following treatments were significantly associated with fertility impairment: busulfan (any dose), carmustine, cyclophosphamide, melphalan (>140 mg/m2) and procarbazine (>5600 mg/m2) [Van den Berg et al. report that the risk of fertility impairment is related to the cyclophosphamide equivalent dose (CED) score, expressing the total alkylating agent exposure. This score reflects the clear dose-effect relationship with alkylating-containing regimens and survivors with a CED score below 7121 mg/m0 mg/m2) .2 (A), docetaxel 75 mg/m2 (T) and cyclophosphamide 500 mg/m2 (C). They reported amenorrhea rates at 12 months of 37.9% for AT, 57.7% for TAC and 69.8% for AC, followed by T [In early breast cancer, cyclophosphamide is one of the cornerstone chemotherapeutics . Patientwed by T . The morwed by T . Dose-dewed by T . In female non-Hodgkin’s lymphoma patients treated with five to six cycles of CHOP, 63% had the return of menstrual cycles 3 months after completing treatment . Others AnthracyclinesAnthracyclines are classified as chemotherapeutic agents with low to intermediate gonadotoxic risk and are widely used in breast cancer, lymphoma and sarcoma treatment ,71. 2, 75 mg/m2 or 100 mg/m2) did report a relationship between cumulative dose  and amenorrhea rates. They reported that in these three treatment groups, 52%, 58%, and 69% of women experienced amenorrhea [Modern breast cancer chemotherapy regimens include anthracyclines. However, their role continues to evolve, mainly due to the risk of cardiotoxicity and secondary leukaemias . The effenorrhea . Early-stage Hodgkin’s lymphoma can be treated with ABVD  . A smallTaxanesThe addition of taxanes in treatment regimens for breast cancer allowed the lower total dose of anthracyclines because of the introduction of sequential regimens .Martin et al. compared TAC with FAC as an adjuvant treatment for early node-positive breast cancer and demonstrated a superior overall survival for TAC. Unfortunately, TAC was associated with a higher rate of amenorrhea compared with FAC (61.7% versus 52.4%) . In addiIn more modern regimens, taxanes are given sequentially rather than concurrently with AC . A smallPlatinum agentsPlatinum drugs appear to have a moderate risk of damaging the ovarian reserve . A smallAntimetabolitesThe use of methotrexate and 5-fluorouracil in breast cancer treatment regimens has not been associated with higher rates of amenorrhea . SantabaMonoclonal antibodiesFor trastuzumab, the risk of gonadotoxicity is considered to be low based on the following data. First, Morarji et al.  report aThe ATEMPT trial compared adjuvant treatment in HER2-positive breast cancer with paclitaxel plus trastuzumab versus TDM-1 . In the TDM-1 group, 75% of premenopausal women had menses at 18 months compared with less than 50% in the control arm, suggesting a potential gonadal safety for TDM-1 .About bevacizumab, a monoclonal antibody against VEGF used in cervical cancer and colon cancer , LorenziRituximab, a monoclonal antibody targeting the CD20 antigen, is widely used in haematological malignancies. The addition of rituximab to chemotherapy did not increase the risk of impaired ovarian function, especially in women younger than 40 years old .Tyrosine kinase inhibitorsRegarding the fertility effects of imatinib (BCR-ABL tyrosine kinase inhibitor), results are scarce for women. There is only one case report of POI in chronic myeloid leukaemia (CML) patients receiving high-dose imatinib . BussiesPazopanib (TKI with anti-angiogenic activity) was also found to induce oligomenorrhoea; however, discontinuation of treatment led to a return of regular menses within 2 months . A cliniOthersRegarding BRAF-MEK inhibitors, protein kinase inhibitors used in BRAF-mutated melanoma, such as dabrafenib and trametinib, their impact on the fertility of women is unknown .Olaparib, a PARP inhibitor, does not appear to interfere with fertility . In addiThe fertility effects of cyclin-dependent kinase (CDK) 4/6 inhibitors remain unknown. These are protein kinase inhibitors involved in cell cycle regulation ,28.There is no evidence for primary hypogonadism in women caused by immunotherapy . In compSpermatogenesis takes place in the seminiferous tubules of the testes, and the required time to develop mature spermatozoa is 74 days. This process begins in puberty and continues throughout adult life. The male primordial germ cells or spermatogonia are located in the seminiferous tubules and are mitotic cells up until they differentiate to initiate meiosis. When meiosis takes place, haploid spermatids are produced, and these differentiate further into spermatozoa. Subsequently, sperm maturation is completed in the epididymis. Sertoli cells, located in the seminiferous tubules supporting the germ cells, play an important role in promoting spermatogenesis in response to testosterone, secreted by Leydig cells and FSH .Semen analysis is the cornerstone of male fertility assessment by evaluating sperm volume, sperm concentration, motility and morphology of the spermatozoids. The impact of systemic oncological therapies on spermatogenesis can vary between azoospermia (absence of sperm cells), oligozoospermia (decreased sperm concentration), asthenozoospermia (reduced sperm motility) or teratozoospermia  . When seIn general, gonadotoxicity is expressed as the risk of developing prolonged azoospermia. It is divided into three classes: high risk, intermediate risk and low to very low risk, with a chance of prolonged azoospermia of more than 75–80%, 25–75% and less than 25%, respectively ,58.Chemotherapy can cross the blood–testis barrier and is known to cause short-term and long-term harmful effects on male fertility . About 2First, infertility can be caused by direct injury to germ cells, depending on the phase of spermatogenesis at the time of drug administration . The proMature sperm cells are more resistant to chemotherapeutic depletion but highly sensitive to DNA damage because spermatids lack DNA repair mechanisms ,95. The Second, impairment of somatic cells and endocrine or paracrine testicular functions can contribute to infertility. Data on the impact of chemotherapy on somatic cells in the testes are limited to Sertoli cells and Leydig cells. Macrophages also play a role in fertility, but their role in gonadotoxicity caused by chemotherapy is not investigated . SertoliNext to Sertoli cell and Leydig cell dysfunction, interstitial fibrosis and reduced blood flow in the testis are described as a consequence of chemotherapy ,99. AnotSemen volume, sperm concentration, sperm motility and sperm viability are semen parameters that are affected by chemotherapeutic agents ,100. ChaAs parallel for women, the c-KIT and PDGFR pathways are important for testis development, hormone production and spermatogenesis. Inhibition of these pathways by TKIs can result in decreased spermatogenesis. PDGFR is expressed by Leydig cells, and blocking the PDGFR pathway by treatment with imatinib can induce apoptosis of these cells, leading to lower testosterone concentrations . Most caAs for women, the risk of gonadotoxicity of ICIs in men remains unclear. IrAEs, such as hypothyroidism or hypophysitis, can cause decreased testosterone levels due to secondary hypogonadism . Unlike The following sections describe the gonadotoxic effects of each class of chemotherapy. Alkylating agentsAlkylating agents have the most detrimental effects on fertility in a dose-dependent manner and are used in AYAs, for example, in sarcoma and lymphoma patients. Cyclophosphamide reduces sperm parameters , decreases testicular weight, and increases sperm abnormalities. In addition, it is associated with increased FSH levels, reduced inhibin B levels and reduced testosterone levels .2 [Sarcoma patients treated with a combination of cyclophosphamide, doxorubicin and dacarbazine with or without vincristine developed azoospermia within 4 months of therapy. Five years after finishing chemotherapy, 40% of patients had normal sperm parameters. The cumulative dose of cyclophosphamide was the major risk factor for gonadotoxicity, with only 10% recovering to normospermic levels when doses exceeded 7.5 g/m2 .In non-Hodgkin’s lymphoma (NHL) patients treated with CHOP, 22% of men were found to be azoospermic 2 years after treatment . PallottIn male Hodgkin’s lymphoma patients treated with BEACOPP, 89% will become azoospermic with higher levels of permanent azoospermia in patients who received 6–8 cycles compared with 2–4 cycles. When 2–4 cycles were used, spermatogenesis could return 3–4 years post-treatment . BEACOPPPlatinum agentsCisplatin is known to decrease sperm parameters and induce alterations of FSH, LH and testosterone. On top, it is associated with a decrease in testicular volume .Platinum agents are the cornerstone of testicular cancer treatment. A study in patients with testicular cancer treated with cisplatin and/or carboplatin described an increase in sperm DNA alterations and aneuploidy at 12 and 24 months after treatment . SantabaAnthracyclinesAnthracyclines cause abnormalities in hormone levels and decrease sperm parameters . In maleTaxanesAs for women, data on the gonadotoxic effects of taxanes are limited. A study of male patients who received taxane-based chemotherapy showed reduced inhibin B levels and elevated FSH levels in all patients during chemotherapy. A significant number of patients had a reduction of testicular volume. No data are available on recovery after treatment .Some data are available on the fertility effects of imatinib. This BCR-ABL tyrosine kinase inhibitor reduces the total number of spermatogonia, but it does not affect spermatogenesis, suggesting that future fertility is preserved. For example, a study in CML patients treated with imatinib showed reduced sperm density, sperm counts, survival rates and activity, and it confirmed that imatinib crosses the blood–testis barrier. However, imatinib did not affect the structure of the testes or the sex hormone levels . In addiA clinical study for gefitinib (EGFR TKI) shows suppression of androgen levels in men and women, but there are no further data on the effect on fertility . ClinicaIn contrast to women, there are some case reports and case series describing orchitis with primary hypogonadism in men treated with ICI ,111. A sAs for women, it is known that ICIs can cause hypophysitis, with the highest incidences for the combination of anti-PD1 and anti-CTLA4 . One casThis review aims to provide oncologists, reproductive endocrinologists and gynaecologists with a practical overview of the current knowledge about the impact of systemic oncological treatments on the fertility of female and male adolescents and young adults. For chemotherapy, the effects on fertility are well described, and it is known that alkylating agents have the most detrimental consequences for female and male fertility. Additionally, other classes of chemotherapy, such as anthracyclines and platinum agents, have an intermediate risk of prolonged amenorrhea in women and permanent azoospermia in men. In general, chemotherapy-induced gonadotoxicity is dose-dependent. For women, the risk of gonadotoxicity is also depending on gonadal reserve before treatment and patient age. Regarding the fertility effects of targeted therapy and immunotherapy, data are too limited to draw conclusions. The potential mechanisms are still under investigation, and there is an important lack of clinical data. Additionally, the effects of each TKI or ICI can be different, so results cannot be extrapolated, and every new treatment needs to be investigated on an individual basis.Measuring the gonadal effect of oncologic treatments is difficult as several tests are available, but none have shown to be the absolute standard for measuring gonadotoxicity. In light of this, studies are heterogenic because of the lack of well-standardized outcomes and definitions of infertility. Some studies report hormonal outcomes, some report ultrasound measurements, and others report clinical outcomes such as loss of menses or azoospermia, which are surrogate measures of infertility. For women with treatment-related amenorrhea, it is important to know that even after the resumption of menses, they can still be at risk for POI. Therefore, it is important to distinguish between short- and long-term results, but studies often lack a prolonged follow-up.Healthcare practitioners may experience difficulties estimating the fertility risk for their cancer patients because of the heterogeneity of study results, patient-specific factors and constantly evolving and heterogeneous treatment regimens. Despite the difficulties in assessing the risk, it is extremely important to adequately inform a patient about the risk of infertility and possible long-term health problems, such as POI. Husson et al. described the wide range of 17% to 83% of AYA patients with haematological malignancies that had a discussion about the potential impact of their treatment on fertility and the possibilities of fertility preservation before initiating treatment. Qualitative research also showed that AYAs reported unrealistic expectations and uncertainties regarding their reproductive potential after treatment which negatively impacted their quality of life . These rNext to assessing and investigating the possible risk of oncological treatments, researchers are looking for possible preventive strategies for reducing chemotherapy-induced gonadotoxicity. One of the most controversial topics is the use of gonadotropin-releasing hormone analogues (GnRHa) for preventing POI. The hypothesis states that downregulation of FSH production can prevent the accelerated primordial follicle recruitment and thus prevent the second hit to the ovaries and preserve the ovarian reserve. Side effects of GnRHa are related to low estradiol levels ,119. DatOther fertoprotective agents under investigation in women are TKIs, such as imatinib, mTOR inhibitors, G-CSF, anti-oxidants, tamoxifen and Sphingosine-1-phosphate . Some prFertility preservation in AYAs with cancer is one of the major issues when dealing with the long-term sequellae of cancer treatment. Timely referrals to oncofertility services to maximize the quality of life of young cancer patients are of utmost importance, and it is essential for physicians to be well-informed about the impact of oncological treatments on gonadal function and fertility, as well as the different fertility preservation techniques. This review provides physicians with a practical overview of the current knowledge about the impact of systemic oncological treatments on the fertility of female and male AYAs, including targeted therapy and immunotherapy. While the effects of chemotherapy on fertility are meanwhile fairly understood, insufficient data are available on the fertility effects of targeted therapy and immunotherapy.This work also underlines the need to further explore the possible harmful effects of systemic oncological treatments on fertility. This unmet need will become increasingly important given the continuously evolving treatment regimens, for example, the introduction of ICIs in the (neo-)adjuvant oncological setting. In this regard, it would be useful to include uniform fertility outcomes as subanalyses in future clinical trials investigating new and existing treatments to determine the long-term fertility effects. Moreover, it would be of great value to track the application and outcomes of fertility preservation strategies in (inter)national registries. Additionally, data relating to AYA patients remain scarce as they are often underrepresented in clinical trials. Not only to make progress in the treatment and survival of their oncological disease but also regarding the topic of fertility, special attention needs to be given to the inclusion of AYAs in future research.


Antibody screening tests for human T-cell leukemia virus type 1 (HTLV-1) are performed based on methods such as chemiluminescent enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay, and particle agglutination (PA). Espline HTLV-I/II, a commercially available, easy-to-use, and rapid immunochromatographic antibody test (IC), was developed for situations where expensive instruments and laboratory equipment are not available. In this report, we compared the performance of IC with the above existing tests using diverse samples derived from asymptomatic HTLV-1 carriers and patients with HTLV-1-associated diseases in collaboration with 11 Japanese institutes. We found that IC detected HTLV-1 infection in all samples from HTLV-1-associated diseases, including adult T-cell leukemia, HTLV-1-associated myelopathy, and HTLV-1 uveitis (200/200). The sensitivity of IC compared with CLIA, CLEIA, and PA was 99.2% (363/366), 100% (241/241), and 100% (47/47), respectively, and the specificity was 99.4% (994/1000), 100% (60/60), and 100% (40/40), respectively. The positive and negative predictive values of IC were 99.7% [95% confidence interval (CI): 99.12–99.92] and 99.5% (95% CI: 98.82–99.75), respectively. However, IC had difficulty in correctly judging samples that were diagnosed as seroreactive in other first screening tests but negative by a confirmatory test; for example, of 612 confirmed negative samples that were CLIA seroreactive, 332 samples were IC positive. These results confirmed that IC has sufficient sensitivity and specificity as a screening test for HTLV-1, although, like the other screening tests, it also requires a confirmatory test to determine HTLV-1 infection correctly.The World Health Organization estimated that 5–10 million people are infected with human T-cell leukemia virus type 1 (HTLV-1). This number is likely to be underestimated because reliable endemic data are available for only approximately 1.5 billion people worldwide. The point-of-care test is a powerful tool for the easy and quick detection of infections without the requirement for expensive instruments and laboratory equipment. Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test that was evaluated in this study, might significantly advance our understanding of the global epidemiology of HTLV-1 infection. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes a life-long infection by integrating its proviral genome into the host genome . The WorHTLV-2 is another retrovirus that is closely related to HTLV-1 . HTLV-1 Diagnostic methods for HTLV-1 or HTLV-1/2 antibody screening such as chemiluminescent enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), and particle agglutination (PA) are available –15. CLA point-of-care test is a powerful tool for the easy and rapid detection of infections without the requirement for expensive instruments and laboratory equipment; however, no serological point-of-care test kits for HTLV-1 and HTLV-2 are commercially available worldwide.in vitro diagnostic kit in Japan since 2023. This kit is based on the detection of a serological reaction between specific human IgG and recombinant HTLV-1 gp21 antigen, and the synthesized peptides HTLV-1 gp46 and HTLV-2 gp46, in a lateral flow assay.Espline HTLV-I/II, an immunochromatographic antibody test (IC), was developed as a novel point-of-care test kit for the diagnosis of HTLV-1 infection and has been authorized as an In this study, we collaborated with 11 Japanese institutes, and collected sera or plasma samples from patients with HTLV-1-associated diseases, outpatients, pregnant women, and voluntary blood donors, and the sensitivity and specificity of Espline HTLV-I/II were evaluated. Moreover, of the collected samples, cases with a discrepancy between screening and confirmatory tests were examined for precise evaluation. Throughout the evaluation, the performance of Espline HTLV-I/II in diverse HTLV-1 diagnostic situations was confirmed.Overall, 2,855 samples  were enrolled. Out of these samples, 1,115 were confirmed positive for HTLV-1 and 1,740 were confirmed negative for HTLV-1. The antibody test methods used in this study are listed in Furthermore, IC correctly detected 100% of WB indeterminate/PCR-positive samples, indicating that IC did not miss these provirus-positive samples as a first screening test. In addition, as shown in Next, the specificity of IC was evaluated using samples that were not reactive at the first screening. Importantly, the results of IC were 100% when using PA and CLEIA, and 99.4% of the results matched those of CLIA  . The senNotably, there was a significant number of inconsistent results for samples that were seroreactive at first screening but negative for LIA  or were LIA indeterminate and PCR negative  .To analyze the inconsistency between the results of IC and the existing kits, we focused on three samples that were HTLV-1 positive and IC negative . The intAn estimated 5–10 million people are infected with HTLV-1, but this number is calculated from limited epidemiological data based on only 1.5 billion people . One limAntigen double sandwich enzyme-linked immunosorbent assay methods for HTLV-1 antibody tests show high performance in terms of HTLV-1 diagnosis. The Architect rHTLV-I/II assay showed 100% sensitivity and 99.98% specificity with previously diagnosed samples, and the results completely matched with the Murex HTLV-I/II test . SimilarIn this study, the performance of a newly developed IC, Espline HTLV-I/II, was evaluated using samples obtained under various conditions during the diagnosis of HTLV-1 infection. In particular, IC had 100% sensitivity for cases with HTLV-1-associated diseases ATL, HAM, and HU. Extremely high sensitivities were also observed for samples from HTLV-1 carriers regardless of the type of screening kit used. Similar high specificity performance was also observed when screening HTLV-1-negative samples. The sensitivity and specificity of IC were thought to be equivalent to the high performance HTLV-1 antibody tests described above. The confidence interval (95%) of IC overlapped with those obtained with the PA, CLEIA, and CLIA methods . These rOur results also suggested the usefulness of IC in clinics or situations where expensive automated instruments for antibody tests are not available. However, IC had a high rate of false positives for samples that were seroreactive at screening but confirmed negative by LIA or PCR. Thus, it is important that diagnoses are not made using IC positive results alone. This concern may be addressed by updating the diagnostic flow of the determination of HTLV-1 infection by incorporating IC as a screening test.HTLV-1 gp21 is a prominent antigen for the detection of HTLV-1 infection , 26. EveBecause the IC detection system is based on an antibody–antigen binding reaction, it might provide a negative result for samples with low antibody titers, such as those obtained at the early stage of infection. As shown in this study, IC did not correctly identify infection in a small number of seropositive samples with antibody titers close to the assay cutoff level. A previous study estimated a window period of at least 2.2 ± 0.6 months for the serological detection of HTLV-1 using HTLV-1 screening tests . AlthougBecause of the limitation of available samples, this study was performed on Japanese samples only. The phylogenetic background of the majority of samples was thought to be subtype A and the transcontinental and Japanese subgroups. The performance of IC with samples from other large endemic areas, such as the regions with subtypes B and D in Africa or subtype C in Australia, and against HTLV-2 should be evaluated further. In addition, because we could only use the first screening kits licensed in Japan, we could not evaluate the performance of IC with other previously reported screening kits. Furthermore, the majority of enrolled samples were screened by CLIA, and the characteristics of the enrolled samples were possibly influenced by the performance of the first screening kit.In conclusion, this study confirmed that Espline HTLV-I/II is a reliable HTLV-1 screening test.The performance of IC was evaluated using the serum or plasma samples that were previously diagnosed by HTLV-1 screening tests and confirmatory tests. Samples were obtained collaboratively from 11 Japanese institutes. The storage temperature of samples was below −20°C. The diagnostic criteria for HTLV-1 infection were based on guidelines for HTLV-1 infection in Japan . PreciseHTLV-1-positive and HTLV-1-negative sera or plasma were obtained with informed consent from patients with HTLV-1-associated diseases including ATL, HAM, and HU, asymptomatic carriers, pregnant women, blood donors, and from residual samples obtained during general clinical practice.Espline HTLV-I/II, an IC for HTLV-I/II, was kindly provided by Fujirebio Inc. . To confirm HTLV-1 infection, initial screening tests were performed by PA, CLIA, or CLEIA . Secondaχ2 test using the GraphPad Prism 7 software . Overall positive and negative productive values of IC were also statistically calculated by bivariate analysis with χ2 test using GraphPad Prism 7.The sensitivities and specificities of IC to other screening tests with a 95% confidence interval were statistically calculated by bivariate analysis with the


The demand for fertility preservation has increased substantially over the past decade as more women wish to delay childbearing and with improved survival outcomes of various medical conditions. This study evaluated the awareness and perceptions of Filipino obstetrician-gynecologists on fertility preservation.A cross-sectional survey was conducted among diplomates and fellows of the Philippine Obstetrical and Gynecological Society from September to December 2021. A self-administered questionnaire with 24 items was distributed online. Univariate descriptive statistics were reported as means for continuous variables and frequencies with percentage for categorical variables. Differences in responses were tested using the chi-square test.A total of 215 respondents completed the survey. Majority of the respondents were female, general obstetrician-gynecologists practicing in the National Capital Region. There was an overall positive perception of fertility preservation, with 98.60% agreeing that discussions about childbearing intentions should be initiated. Most participants (98.60%) were aware of fertility preservation but had varying levels of awareness of the different techniques. Fifty-nine percent of the respondents were unaware of regulations on fertility preservation. Setting up dedicated centers for fertility preservation and offering it as a public service were viewed as necessary by the respondents.This study underscored the need to increase awareness of fertility preservation techniques among Filipino obstetrician-gynecologists. Meeting the need for comprehensive guidelines and centers is essential to promote fertility preservation in the country. Efficient referral systems and multidisciplinary approaches should be established for holistic care. One of the most critical milestones in reproductive medicine is the advent of fertility preservation. Various fertility preservation techniques allow men and women with compromised fertility a chance to achieve reproductive capacity at a later time. While advances in cancer therapy have led to an increasing number of young patients who survive, a crucial sequela is loss of fertility due to the gonadotoxic profile of current regimens . The fieAge is a critical factor in the Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) for women with poor ovarian response to stimulation . Age dirThe Philippine Society for Fertility Preservation was established in 2019, reflecting the growing need and interest to improve and promote its practice. Awareness of established fertility preservation techniques is essential to ensure appropriate counseling of patients and referral to a specialist. Currently available fertility preservation strategies in females include embryo cryopreservation, mature oocyte cryopreservation, ovarian tissue cryopreservation, ovarian suppression with GnRH analogs, ovarian transposition, and fertility-sparing surgeries [In the Philippines, fertility preservation techniques are only offered in private centers and paid through out-of-pocket expenses. At the time of writing, no government-funded facility offer these procedures. Expenses are not covered by the Philippine Health Insurance Corporation nor by health maintenance organizations.Despite the rapid progress of fertility preservation in clinical practice, knowledge of its availability is lacking among clinicians . This paA cross-sectional survey was conducted among diplomates and fellows of the Philippine Obstetrical and Gynecological Society (POGS) from September to December 2021. A hyperlink to the online survey was sent by electronic mail to the target participants with society’s support and distributed over social media. The minimum required sample was 209 of the 4500 accredited obstetrician-gynecologists in the country. This was computed using the Cochran formula and based on the study by Fritz et al. , which rA self-administered survey patterned from the study of Chung et al. . was utiP value of < 0.05 was considered statistically significant. All analyses used STATA 14 (Stata Corp Inc).Univariate descriptive statistics were reported as mean for continuous variables and frequency with percentage for categorical variables. Differences in responses were tested using the chi-square test. A A total of 215 participants accomplished the online questionnaire. The mean age of the respondents was 42.98 ± 10.59 years. The majority were female (94.88%), Catholic (80.94%), married (68.37%), and had children (65.12%). The geographical regions were represented, with the National Capital Region (NCR) being the most represented. The sociodemographic data of the respondents are summarized in Table Most of the participants belonged to private non-university affiliated hospitals 27.91%) and had practiced for one to five years (44.65%). General obstetrician-gynecologists constituted 57.21% of the study population. Of the 42.79% specialists, the most frequently identified specialties were Ultrasound (12.56%) and Reproductive Endocrinology (10.70%). Table 7.91% andMajority of the respondents agreed that obstetrician-gynecologists should initiate discussions with patients about their childbearing intentions (98.60%) and age-related fertility decline (97.67%). Obstetrician-gynecologists largely believed that discussion of natural fertility decline should be part of a well-woman annual examination, with agreement by 96.28%.Almost all participants (98.60%) were aware of fertility preservation and were familiar with at least one method or procedure. Only 32.56% were familiar with all techniques, including fertility-sparing surgeries, the use of GnRH agonists, sperm freezing, oocyte freezing, embryo freezing, and ovarian or testicular tissue freezing. Most respondents (81.40%) were aware of fertility-sparing surgeries. Approximately half (45.12%) of the participants have not referred patients for fertility preservation in the twelve months before the study proper. Only seven respondents were able to refer patients for all the mentioned procedures (Table Respondents were largely aware (86.98%) of a particular clinic or specialist who can accept referrals for fertility preservation. Of note, 28 respondents (13.02%) were unaware of any facility or specialist. In 43.72%, the patient’s desire to have children was identified as the most critical factor when deciding on fertility preservation in medical indications, followed by age (35.81%) and prognosis (10.23%).Majority of the participants 93.49%) deemed it necessary to set up dedicated centers for fertility preservation. About 91.63% think it should be offered as a public health service. Standard educational materials were deemed essential in enhancing patient understanding of fertility preservation. More than half (59.07%) are unaware of regulations relating to fertility preservation, but 98.14% support establishing guidelines. Three respondents did not wish to know more about fertility preservation  is to promote the science and practice of fertility preservation. The society conducts regular conferences, meetings, and discussions on scientific information and treatment advances. There were varying levels of awareness of the different techniques. Most were familiar with at least one fertility preservation technique. Meanwhile, only a third of the study population knew all methods. Unawareness may lead to the underutilization of available methods of fertility preservation. This emphasizes the need to educate more obstetrician-gynecologists through fertility preservation awareness campaigns and continuing medical education activities, including seminars and workshops.Not surprisingly, the highest level of awareness was associated with fertility-sparing surgeries. Fertility-sparing surgery entails preserving at least a portion of an ovary and the uterus. These are limited to early-stage malignancies and include conization or trachelectomy for cervical cancer and unilateral salpingo-oophorectomy for ovarian cancer. Clinicians should provide appropriate information about oncologic and pregnancy outcomes through an individualized patient approach . ObstetrDespite the high level of awareness, half of the respondents had not referred patients for fertility preservation, and majority desired to know more information. The study’s findings were similar to the reports of Harzif et al. among obstetrician-gynecologists in Indonesia . IdentifMost respondents saw setting up dedicated centers for fertility preservation as necessary. The study shows that reproductive endocrinologists have 80% lower odds of agreeing on this than general obstetrician-gynecologists. A small proportion of the study population was unaware of any facility or specialist. In the Philippines, fertility preservation techniques are mainly performed in reproductive centers offering in vitro fertilization. There are only eight centers and 147 infertility specialists able to provide these services in the country. Access to these centers is available to reproductive endocrinologists, which may explain the decreased support for establishing dedicated facilities. Encompassing help from all specialists should be elicited to promote fertility preservation.Early referral of women with malignancy at the time of diagnosis and before treatment commencement is the key to maximizing the success of fertility preservation and allows a greater window of opportunity for preserving fertility . As primThe high cost of most fertility preservation techniques and patient financial constraints have impeded widespread local use. Most respondents agreed that these techniques should be offered as a public health service to mitigate access issues. A multilevel approach is essential to address issues specific to patients and their families, clinicians, organizations, policymakers, and the general population .Fertility preservation is a significant issue in women diagnosed with malignancy. A survey of young women undergoing therapy showed that childbearing remains a priority . DiminisComprehensive recommendations and clinical guidelines on fertility preservation should be established and communicated. ESHRE published its first evidence-based guideline on female fertility preservation for healthcare professionals in 2020 . Socio-eOverall, Filipino obstetrician-gynecologists have an encouraging positive perception of fertility preservation. There is a need for further education on the locally available techniques. The information presented by this study can be applied in the framework of establishing local guidelines and designing a curriculum for training. A multidisciplinary team with reproductive specialists, an insurance coverage system, comprehensive laws, and practice guidelines should be prioritized.Reproductive aging and fertility preservation are emerging fields in managing reproductive-aged women. This is the first local study that evaluated the awareness and perceptions of post-residency obstetrician-gynecologists on fertility preservation. The study showed a reassuring positive perception of fertility preservation but a gap in the awareness of different approved methods. A multidisciplinary approach and dedicated facilities should be established for fertility consultation, risk assessment, and counseling. Healthcare delivery should be organized to meet the increasing need for fertility preservation.The study employed non-probability sampling and consecutive enrolment of participants until the sample size was met. Selection and response bias may have influenced the results of the study. Participation in a self-directed online questionnaire entails the awareness of the sample to the existence of the survey. The number of physicians who actually received the survey is uncertain. This was minimized by distribution of the questionnaire by the Philippine Obstetrical and Gynecological Society to its registered members. Regular posting of the survey to various social media platforms was also conducted to improve visibility and response. To minimize response bias, the period of data collection was extended after the minimum sample size was met. The number of Gynecologic Oncology specialists who completed the survey was only eight due to the sampling method employed. The study did not assess the specific reasons for non-referral for fertility preservation techniques.Further studies with the recruitment of gynecologic oncologists may be undertaken. Another vital area of research is the investigation of perceived barriers to the provision of timely and appropriate fertility preservation techniques. The knowledge and perceptions of patients on fertility preservation should also be investigated.


Human T-lymphotropic virus 1 (HTLV-1) infected individuals remain as asymptomatic carriers (ACs) or can develop the chronic neurological disorder HTLV-1-associated myelopathy/Tropical Spastic Paraparesis (HAM/TSP) or the adult T-cell leukemia/lymphoma (ATLL), and the immunological mechanisms involved in this pathologies need to be elucidated. Recently, it has been demonstrated that induced or naturally developed IgG repertoires obtained from different groups of donors, grouped by immune status, can modulate human T and B cell functions. Here we aimed to evaluate if the IgG obtained from HTLV-1-infected ACs, HAM/TSP, and ATLL patients can differentially modulate the production of cytokines by human T and B cells. With this purpose, we cultured PBMCs with IgG purified from ACs, HAM/TSP, or ATLL donors and evaluated the frequency and intracellular cytokine production by flow cytometry. Our results indicate that IgG from HAM/TSP patients could induce an augment of IL-17-producing CD4+ T cells, reduce the frequency of IL-4-producing CD4+ T cells, increase IFN-γ-producing CD8+ T cells, and reduce IL-4-producing CD8+ T cells. IgG from ATLL could reduce the frequency of IL-4-producing CD4+ T cells, similarly to IgG from HAM/TSP /TSP, and could reduce the frequency of IFN-γ-producing γδT cells without influence on IL-17- and IL4-producing γδT and could reduce the frequency of IL-10- producing B cells. Finally, IgG from both HAM/TSP and ATLL patients could reduce the frequency of IFN-γ producing B cells. In conclusion, these results suggest that these preparations are active, partly overlapping in their effects, and able to elicit distinct effects on target populations. HTLV-1 is the retrovirus responsible for HAM/TSP and ATLL . More frper se, mediate immune modulation in murine and human lymphocytes  in their plasma , 29. It The control of HTLV-1 proliferation in infected individuals is dependent on the induction of effector immune mechanisms, including the production of IgG antibodies that recognize the virus and stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells . ImmunolBased on those observations, in the present study, we aimed to evaluate the effect of the IgG repertoires obtained from asymptomatic, HAM/TSP, and ATLL donors on the production of cytokines by peripheral αβT- (CD4+ and CD8+), γδT- and B-cells searching for the identification of differential immunological signatures that may be related to each clinical manifestations resultant from HTLV-1 infection.n = 14; 12 males and 2 females; Age: 52.7 ± 2.7; Pro-viral load median: 13 copies/1000 PBMCs), HAM/TSP , and ATLL  patients. HTLV-1-positive individuals were recruited from the HTLV-1 outpatient clinic at the University of São Paulo and the Institute of Infectious Diseases “Emilio Ribas.” All ACs were diagnosed as HTLV-1 carriers at the time of blood donation. Viral infection was identified by the Murex HTLV I + II  and Vironostika HTLVI/II  HTLV enzyme immunoassays, and infection was confirmed by HTLV BLOT 2.4 . The clinical status of HAM/TSP was determined based on the WHO criteria for HTLV-1-associated diseases (n = 30) or to obtain the PBMCs (n = 10) used in the culture experiments avoiding autologous IgG-PBMCs experiments. Written informed consent was obtained from each participant. The study was approved by the local review board.Purified IgG was obtained from randomly selected patients from a larger cohort of 233 HTLV-1-infected persons representing asymptomatic carriers . Purified IgG was collected, sterilized using 0.20-micron filters , and stored at −80°C for cell culture experiments. Following the manufacturer’s instructions, IgG concentrations were determined using Coomassie Protein Assay Reagent . The purity of IgG, as evaluated by SDS-PAGE, was greater than 95%, and the 5% contaminants had molecular weight lower than 10 kDa indicating that they are predominantly protein fragments that the melon gel could not capture and that they probably do not include molecules of biological importance such as cytokines. All pools were evaluated, and undetectable IgA, IgM, and IgE antibody levels were confirmed. The frequency of IgG subclasses, as evaluated by ELISA, was similar between all IgG formulations. This purification protocol was evaluated for the quality of the purified IgG, and it demonstrated higher efficiency in purifying idiotype-functional molecules and avoiding complex IgG molecules . Further6 viable PBMCs were placed in each well of a 96-well culture plate  and cultured with 100 μg/mL IgG purified from pooled serum samples from each group of patients or controls in RPMI 1640 medium containing 10% FC-III . The mock condition or the addition of 100 μg/mL commercially purified IgG (IVIg) was used as an additional control. The culture plates were incubated for 3 days, and 1 μg/mL brefeldin A  was added in the last 12 h as previously standardized . Using a Neubauer chamber, a cell suspension aliquot was diluted in trypan blue  to evaluate the cell viability and number. Then, 1 × 10dardized , 24, 37.Cell culture and flow cytometry were performed using protocols that were previously described by our group , 38, 39.in vitro studies were taken from 5 separate experiments with 2 PBMC donors per experiment. Differences were considered significant at p ≤ 0.05 as assessed by Student’s t-test and the Mann–Whitney U test (comparisons between two groups) or by an ANOVA test with Tukey’s test correction (comparisons among more than two conditions) because of the Gaussian distribution of the values.Statistical analysis was performed with GraphPad Prism 5.0 . Data from To evaluate the effects of grouped IgG idiotypes repertoires, we purified and pooled IgG antibodies from ACs, HAM/TSP, and ATLL patients and performed culture experiments using PBMC from healthy non-infected individuals. As control conditions, all experiments included the mock condition (absence of IgG), the IVIg condition , and the HCs condition .After 3 days of culture, all evaluated culture conditions did not influence the frequency of CD4+ T cells , and a sWhen evaluating γδT cells, we could not detect any differences in the frequency of these cells between culture conditions . OtherwiUltimately, we also evaluated the frequency of B cells after the culture period, and we could not detect any differences in the frequency of these cells between culture conditions . By evalin vivo conditions, the IgG antibodies may collaborate, triggering an IFN-γ-mediated response and HTLV-1-related symptoms and favoring HAM/TSP development, an aspect that needs further elucidation.HAM/TSP is potentially considered an immune-mediated disease, mainly due to impaired production of cytokines, including IFN-γ levels . Our obsOf note, our results also indicated that IgG from HAM/TSP patients could induce a higher frequency of IL-17-producing CD4+ T cells (Th17) without influencing the production of this cytokine in all other evaluated populations. This evidence corroborates with the literature that demonstrated increased plasma levels of IL-17 in HAM/TSP patients  since Thin vitro , which we intend to evaluate in future studies. Although, our results indicate that the IgG repertoire may, at some point, influence the cellular immune response profile in biomarkers that can influence HTLV-I pathogenesis. This aspect was scarcely discussed in the literature, but it was demonstrated 20 years ago that IgG isolated from HAM/TSP patients could recognize the human heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1). This molecule could be listed as a target molecule mediating HAM/TSP development . In the In conclusion, our observations could demonstrate that different populations of peripheral T  and B cells can differentially produce IFN-γ, IL-4 IL-10, and IL-17 according to the repertoire of IgG produced by each group of HTLV-1-infected individuals , what may collaborate on framing the control or development of disease. These observations open a new field of investigation that may collaborate with elucidating HTLV-1 infection pathogenesis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by The study was approved by the review board of the University of São Paulo, Medical School, São Paulo, Brazil. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.in vitro experiments and data analysis. AO, YN, and JC carried out the diagnosis and selection of patients. FC performed in vitro experiments. LN and SS critically revised the manuscript. JV coordinated the activities of all other collaborators, obtained funding to carry out the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.NM, BF, and LF performed This study was funded by grants from the Laboratory of Medical Investigation-56, Medical School, University of São Paulo, São Paulo, Brazil (LIM-56 HC-FMUSP), National Council for Scientific and Technological Development (CNPq) grant# 302937/2021-8 and São Paulo Research Foundation (FAPESP) grant #2021/08225-8.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Functional neurological disorder (FND) presents motor, sensory, and cognitive symptoms characterized by clinical signs incongruent with known neurological disease. Together with other health professionals, like neurologists, psychiatrists can play an essential role in diagnosing and managing these disorders. Hence, understanding their opinion and clinical experience with FND is of utmost importance to catch potential educational needs and improve healthcare services for patients. This study aims at assessing the knowledge, opinion, and clinical approach of Italian psychiatrists to FND.Members of the Italian Society of Psychiatry completed a 14-item web-based survey investigating their approach to FND. Results. Overall, 174 questionnaires were completed. Our main findings suggest that Italian psychiatrists have a psychogenetic conceptualization of FND. “Conversion disorders”, in fact, is the term most frequently used by Italian psychiatrists to refer to FND, thus implying a psychological etiology of these disorders. Congruently with this view, psychotherapy associated with pharmacological therapy is considered the most appropriate treatment by psychiatrists, while physiotherapy is an under-recognized treatment option for FND.The present study highlights that a psychogenetic view of FND dominates among Italian psychiatrists. This could be due to out-of-date knowledge about the pathophysiology of this group of disorders. Promoting education about novel approaches to FND would be of crucial importance to improving care for patients suffering from this condition. Sincsymptoms . CongrueResearch on the pathophysiology of FND has recently challenged such a perspective, moving FND away from a psychogenic conceptualization toward a biopsychosocial model , 5. The The novel diagnostic approach to FND better fits with the expertise of neurologists, which are more trained than psychiatrists in physical examination and classification of neurological disease . NonetheDespite their potential role in improving the diagnosis and treatment of FND, little is known about the opinions and clinical experiences of psychiatrists regarding these disorders. A recent study by Dent et al.  found th2.2.1.The survey was based on the methodology used in our previous study conducted on a sample of Italian neurologists . The queN = 690) by the General Secretary of the SIP using the 2020 members list. The e-mail explained that the survey was meant to investigate opinions, knowledge, and clinical experience with non-organic neurological disorders among psychiatrists. As in our previous studies . The invitation to participate was sent by e-mail to all potential respondents  was used to collect responses over a period of 8 weeks (1 February – 29 March 2022). Two e-mail reminders were sent to the SIP members, 2 and 6 weeks after the initial mailing. The study received ethical approval from the University of Verona (CARP) and was conducted in accordance with the Declaration of Helsinki.2.2.p < 0.05. All the analyzes were performed using SPSS software (version 19).Survey responses were examined with descriptive statistics, including frequencies and percentages. Chi-squared test was used to analyze gender distribution. Statistical significance was set at 3.3.1.p = 0.22) completed the questionnaire  while the remaining were residents . Most were employed in the public sector, either in a community mental health center  or in an inpatient psychiatric unit .Overall, 174 out of 690 contacted psychiatrists , 48 ± 14; mean years of practice ± SD, 18 ± 14), with a balanced number of males (55%) and females (45%)  stated that less than 10% of the patients seen in a week presented with FND. Thirty-eight percent (n = 66) reported a higher proportion of FND (10–25%) and a few  stated that 25–50% of their patients have a diagnosis of FND. Very few  stated that more than half of their patients have FND or were unable to estimate how many of their patients might have a FND  was the most frequently used term to describe FND, followed by “Somatization disorders” , “Functional Neurological Disorders” , and “Psychogenic disorder” . Less frequently used terms were: “Non-organic disorder” , “Unspecific anxious syndrome” , “Hysteria” , “Stress-related disorder” , “Depression” , and “Medically unexplained disorder” . Very few chose the “I do not know” answer .Respondents could choose from a list of 10 terms they usually used to describe functional neurological symptoms . Sixty-o3.2.3.n = 123, 71%) or moderately probable  in this kind of disorder, while 12% (n = 21) found it not at all probable, and very few stated that simulation was highly probable .Most respondents believed that simulation  was little . Many chose “disorder due to abnormal functioning of the nervous system” , while few chose “absent neurological disorder”  or “stress” . Other explanations were: “somatization,” “physical symptoms of emotional distress,” and “poor diagnostic investigation.”When asked about their preferred way to explain symptoms to patients with FND, the majority chose “psychogenic disorder”  reported that “reduction in symptoms with distractive maneuvers” represents a predictive feature of this group of disorders, followed by “normal or inconclusive neurological examination findings”  and “inconsistency” of symptoms  , “other medically unexplained symptoms” , “litigation” , “greater loss of function or disability than found on physical examination” , “previous mental illness or psychological stress”  were considered as poorly predictive , most respondents , and “psychiatric consultation”  were frequently rated as “a lot” or “very much” adequate for FND. On the other hand, “physiotherapist consultation”  and “neurological consultation”  were prevalently rated as “only a little” or “to some extent” adequate for FND patients , “psychotherapy consultation”  was most frequently rated as “a lot” or “very much” appropriate for FND patients , “psychotherapy without antidepressant or anxiolytic medications” , “rehabilitation ” , and “educational interventions”  were frequently rated as “only a little” or “to some extent” adequate.When psychiatrists were asked to indicate the degree of adequacy of five different treatments for FND, “psychotherapy with antidepressant or anxiolytic medications” , and to a “psychologist or psychotherapist” ; more than half of the sample reported that the most appropriate strategy would be “wait and see how symptoms develop” , and to prescribe “instrumental examinations” such as magnetic resonance imaging . The management strategy most frequently excluded from clinical practice was “referral to a physiotherapist” . Half of the sample was undecided about “Pharmacological prescription”  with management strategies, most respondents “agreed” or “totally agreed” that patients with FND should be referred to a “neurologist”  rated their level of satisfaction between 5 and 10, with an overall average score of 5.91 .When asked to rate their satisfaction in managing FND on an 11-point scale from 0  to 10 (extremely satisfied), most respondents  gave more than one response, with “following-up the treatment together with other specialists ” as the most frequent , followed by “educational intervention for patients and their families” , and “make a diagnosis and recommend adequate treatment ” . Nineteen percent (n = 33) of the sample reported that psychiatrists should “make a diagnosis and personally follow-up the patient,” while very few  thought that the role of psychiatrist should be simply to refer FND patients to a specialist for their medical condition.When asked about the clinical role of psychiatrists in the management of patients with FND, most of respondents  of respondents hypothesize that symptoms might be feigned  with a little, moderate, or high probability. Similar results emerged also from our previous study involving neurologists  and migh% of respad hoc neurological examination. This is in line with current guidelines, suggesting that neurological assessment is needed for establishing a diagnosis of FND (In line with the current knowledge of the disease, psychiatrists believe that changes in symptoms with distractive maneuvers and inconsistency over time are the most predictive factors for a diagnosis of FND. These findings suggest that psychiatrists can recognize the typical signs of FND symptoms. However, as a first step in the management of FND, most respondents would ask for a neurological consultation. This approach might be driven by the need for excluding other neurological conditions by means of s of FND . Neurolos of FND , 46. ThuHowever, comparing the current survey with the previous one conducted on neurologists  it seemsSome limitations need to be acknowledged. It is possible that only psychiatrists interested in FND responded to the survey, thus reducing the generalizability of our findings. Furthermore, the limited number of questions might have precluded a more comprehensive understanding of the respondents’ attitudes and knowledge about FND. These limitations notwithstanding, this survey provided novel insights on the knowledge, opinion, and clinical experience of a sample of Italian psychiatrists with FND. Together with our previous studies involving general practitioners and neurologists , 19, theThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involving human participants was reviewed and approved by CARP - University of Verona. The participants provided their written informed consent to participate in this study.MT, AM, and MF: conceptualization and methodology. AM, AL, EZ, GDS, CP, and CB: recruitment of participants and data collection. AM: data curation and visualization. AM, MT, MF, AL, CB, and DP: analysis and interpretation of data. AM: writing – original manuscript. AM, MT, MF, AL, CB, DP, GDS, CP, and CB: writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Urinalysis (UA) results are used by clinicians and laboratories to aid in the diagnosis of UTI. However, optimal UA parameters have not been systematically investigated or validated for different populations. Our objectives were to assess the performance of UA in predicting UTI and to stratify UA performance by age, sex, and culture thresholds.We conducted a retrospective cohort study of adult non-catheterized inpatient and ED encounters with paired UA and urine cultures (24 hours apart) from 5 community and academic hospitals in three states  between 01/01/2017 and 12/31/2019. Trained abstractors collected clinical and demographic data for a random sample of patients. Patients were classified as having UTI, asymptomatic bacteriuria or ‘not UTI’ based on IDSA guidelines using signs/symptoms and microbiology data. We evaluated the performance of relevant UA parameters in predicting UTI by assessing sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). We also combined 18 different UA criteria  and used area under receiver operating curves (AUROC) to identify the 5 best-performing models, and stratified results by age, sex, and lower urine culture bacterial threshold (1000 colony forming units).Of 219,338 encounters, 3392 charts were included and reviewed; 723 (21.3%) patients met criteria for UTI. Females and older adults had a higher incidence of UTIs and ASB . Absence of pyuria (or leukocyte esterase) had a high NPV for UTI (Table 2). Combined UA parameters performed better than pyuria alone with regards to NPV and AUROC, specifically models 1 and 5 (Table 3). UA parameters used in these models performed differently based on age, sex and urine culture thresholds, with limited utility in older women and UTIs with lower bacterial threshold (Table 4).Our review of laboratory and symptom data from a diverse population dataset revealed that combined UA parameters were better at predicting UTI, but performance of UA parameters differs based on age, sex, and urine culture thresholds. Our approach highlights the need to move away from a one-size fits all approach to using population specific UA cut-offs for patients with UTI symptoms.Sonali Advani, MBBS, MPH, FIDSA, bioMérieux: Advisor/Consultant|GlaxoSmithKline: Advisor/Consultant Nicholas A. Turner, MD, MHSc, PDI: Research contract to assess efficacy of cleaning agent


Chimeric antigen receptor (CAR) costimulatory domains derived from native immune receptors steer the phenotypic output of therapeutic T cells. We constructed a library of CARs containing ~2,300 synthetic costimulatory domains, built from combinations of 13 signaling motifs. These CARs promoted diverse cell fates, which were sensitive to motif combinations and configurations. Neural networks trained to decode the combinatorial grammar of CAR signaling motifs allowed extraction of key design rules. For example, non-native combinations of motifs which bind tumor necrosis factor receptor-associated factors (TRAFs) and phospholipase C gamma 1 (PLCγ1) enhanced cytotoxicity and stemness associated with effective tumor killing. Thus, libraries built from minimal building blocks of signaling, combined with machine learning, can efficiently guide engineering of receptors with desired phenotypes. Chimeric antigen receptors (CARs) have demonstrated the power of synthetic signaling receptors as tools to reprogram immune cells to execute therapeutic functions, such as targeted killing of tumor cells . The antA major and still mostly outstanding goal in synthetic biology is to predictably generate new cell phenotypes by altering receptor composition. For example, in cancer immunotherapy, a general goal is to enhance T cell anti-tumor cytotoxicity but to also maintain a stem-like state associated with longer-term T cell persistence. Such a phenotype is associated with effective and durable tumor clearance (higher stemness is correlated with more resistance to T cell exhaustion). Libraries of costimulatory domains have been screened for improved phenotypes –16. HoweWe recombined 13 signaling motifs (words) to create a CAR costimulatory domain library with randomized motif combinations (new sentences) . This lith component in the library. The combinatorial library was constructed within the context of an anti-CD19 CAR . The synthetic costimulatory domains had either one, two, or three signaling motifs. The 13 motifs were randomly inserted in positions i, j, and k   and primj, and k  to yieldj, and k . All T c+ and CD8+ CAR T cell populations , stemness  and killer cell lectin receptor G1 (KLRG1−)) ) , and maiThe CARs in the arrayed screen displayed a range of cytotoxicity and stemness. The total naïve and central memory population was positively correlated with cytotoxicity . Stemnes2 values of approximately 0.7 to 0.9.The diverse cytotoxicity and stemness profiles observed in our arrayed screen are consistent with a complex relationship between signaling motif combinations and arrangement, and resulting T cell phenotypes. We sought to leverage the combinatorial nature of the costimulatory domain library by using machine learning to decode the “language” of signaling motifs that relates motif combinations to cytotoxicity and stemness outputs. We separated the arrayed screen data into a training set (221 examples) and a test set (25 examples). We then used these data sets to train several machine learning algorithms to predict cytotoxicity and stemness based on costimulatory domain identity and arrangement . Neural The trained neural networks then allowed us to predict the CAR T cell cytotoxicity and stemness that would result from each of the 2379 motif combinations in the full combinatorial library , includiTo assess the overall contribution of individual motifs, we ranked all the CARs in our library by neural network-predicted cytotoxicity and stemness and then assessed whether motifs were enriched in the strong or weak ends of the phenotypic distribution , S4. If th percentile) and high stemness (mean 63rd percentile), indicating that overall it promotes both of these phenotypes. In a contrasting example, M6 (from LAIR1) recruits the phosphatase SHP-1, an inhibitor of T cell activation. Accordingly, M6 is enriched in CARs with low cytotoxicity (mean 36th percentile) and low stemness (mean 45th percentile), indicative of inhibition of both phenotypes. Some motifs can activate one phenotype and inhibit another: M5, which binds Vav1, is unrepresented in CARs with high cytotoxicity (mean 25th percentile), but overrepresented in CARs with high stemness (mean 64th percentile). Thus, Vav1 signaling appears to promote stemness, while inhibiting killing. The quantified effects of all individual motifs on cytotoxicity and stemness are shown in the heatmap in This distribution analysis highlighted several effective motifs that have activating and inhibitory roles. For example, motif M9 is the PQVE motif (from cluster of differentiation 40 (CD40)), which binds TRAF2, and is associated with T cell activation and function , 25. Accth and 60th), but the M1+M10 motif pair was even more strongly activating (mean: 75th percentile). The predicted mean cytotoxicity and stemness percentiles for all 144 pairs of motifs M1 to M12 are shown in We anticipated that phenotypes would be highly dependent on motif combinations, as different downstream signaling pathways could be either complementary, redundant, or competing. To examine motif pairs that favored particular phenotypes, we examined the occurrence of each possible pair (without regard to order) in the ranked distribution. Several specific motif pairs appear to promote both cytotoxicity and stemness when they occur in combination within a costimulatory domain. For example, M1 (PLCγ1) and M10 (TRAF) were each activating with respect to cytotoxicity , motif pairs (word combinations), and motif position (word order) on phenotype. The analysis also yields design rules that can inform combinations and arrangements of motifs capable of producing a desired cell fate. For example, a synthetic costimulatory domain that contains one or more TRAF binding motifs (M9 or M10) followed by a PLCγ1 (M1) motif appears to be effective at promoting both cytotoxicity and stemness . AlthougWe examined the neural network-predicted library to predict the effects of adding the M1 motif to CD28-like and 4-1BB-like synthetic costimulatory domains  . The 4-1To experimentally test this, we synthesized derivatives of the 4-1BB and CD28 costimulatory domains with 1 or 2 copies of the M1 motif added to the C-terminus, and tested the effects of these costimulatory domains on killing of Nalm 6 and maintenance of T cell stemness . Consist2), which activates Ras guanyl-releasing protein (RasGRP) and protein kinase C theta (PKCθ), subsequently activating the extracellular signal-regulated kinases (ERK1 and 2) and NFκB. This signaling is similar and possibly redundant to that of PI3K and Grb2, which also activate RasGRP and PKCθ. TRAF signaling, however, does not activate RasGRP or PKCθ, such that PLCγ1 and TRAF signaling are more likely to be complementary (Why might a PLCγ1 motif improve T cell phenotype in combination with the 4-1BB domain (TRAF motifs) but not in the context of the CD28 domain ? PLCγ1 catalyzes the production of diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate , effectively using naturally occurring signaling sentences (motif combinations). We used motifs from receptors as words to generate thousands of different signaling sentences that drove T cells to distinct cell fates, potentially yielding more diverse and nuanced phenotypic meaning. Augmenting experimental analysis of a subset of receptors with neural network analysis allowed us to explore a larger region of combinatorial motif space. In particular, we identified the non-natural combination of TRAF- and PLCγ1-binding motifs that may be useful in CAR T cell therapies. With an arrayed screen of several hundred receptors and machine learning, we identified basic elements of signaling motif language and extracted design rules that relate motif combinations to cell fate. This represents a step toward forward engineering receptors with desired properties. Similar screening approaches with other CARs and target cancer cells are needed to determine the optimal signaling domains for each CAR and tumor type. Libraries may also be of use in identifying combinations of binding, hinge, linker, transmembrane, and signaling domains that produce optimal T cell function, and assessing the safety and toxicity of such combinations. Exploration of these larger libraries may benefit from machine learning due to the size and complexity of the combinatorial space. Machine learning-augmented screens of this type might be used to engineer many other classes of receptors for biological research and cell therapy applications involving cellular processes controlled by combinations of signaling motifs.S1S3S24


Osteoarthritis (OA) is a prevalent musculoskeletal disease that affects approximately 500 million people worldwide. Unfortunately, there is currently no effective treatment available to stop or delay the degenerative progression of joint disease. Wnt signaling pathways play fundamental roles in the regulation of growth, development, and homeostasis of articular cartilage. This review aims to summarize the role of Wnt pathways in joint development during embryonic stages and in cartilage maintenance throughout adult life. Specifically, we focus on aberrant mechanical loading and inflammation as major players in OA progression. Excessive mechanical load activates Wnt pathway in chondrocytes, resulting in chondrocyte apoptosis, matrix destruction and other osteoarthritis-related changes. Additionally, we discuss emerging Wnt-related modulators and present an overview of emerging treatments of OA targeting Wnt signaling. Ultimately, this review provides valuable insights towards discovering new drugs or gene therapies targeting Wnt signaling pathway for diagnosing and treating osteoarthritis and other degenerative joint diseases. Articular cartilage (AC) primarily consists of chondrocytes and extracellular matrix. It provides the joint with a low-friction and wear-resistant surface, enabling it to withstand various mechanical loadings during movement, such as shear stress, hydrostatic pressure, osmotic pressure, tensile force, and pressure stress. Mechanical loading has a “double-edged sword” effect on the growth and function of chondrocyte . During 2+ pathway  and low-density lipoprotein receptor-related receptor 5/6 (LRP5/6), activated Fzd acts on the cytoplasmic scatter protein Dishevelled (Dsh/Dvl), destabilizing the APC/Axin/GSK-3β destruction complex and reducing its phosphorylation level of β-catenin. This process leads to an accumulation of free β-catenin in the cytoplasm, which then translocates into the nucleus and binds to TCF/LEF family members to regulate target gene expression of the Wnt signaling pathway.2+ pathways, are initiated by a variety of Wnt ligands such as Wnt1, Wnt3a, Wnt4, Wnt5, and Wnt11, and Wnt16. These pathways play crucial roles in processes such as cytoskeletal reorganization, chondrocyte stacking, and various phenotypic responses . PLC then catalyzes the production of inositol triphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate in the cell membrane. IP3 releases stored intracellular calcium, resulting in the activation of calcium/calmodulin-dependent protein kinase II (CamKII) and NFAT, a transcription factor that controls cell adhesion and cell migration, as well as protein kinase C (PKC). DAG also activates PKC and mitogen-activated protein kinase (MAPK), which are involved in controlling diverse cellular activities.Non-canonical Wnt signaling pathways, which encompass the Wnt/PCP and Wnt/Caesponses . Wnt/PCPAccumulative studies have provided substantial evidence supporting the crucial role of Wnt signaling pathways in joint development and cartilage homeostasis  Table 1Table 1. The Wnt signaling pathway is essential for joint initiation. On one hand, Wnts can inhibit chondrogenesis by downregulating the expression of Sox9 and Noggin, which promotes the de-differentiation of chondrocyte into interzone cells . On the During joint development, the Wnt signaling pathways crosstalk with other pathways, such as the TGF-β superfamily and Ihh signaling pathway to determine chondrocyte fate and maintain joint homeostasis maintenance . The groIn a study of Wnt signaling using transgenic mice carrying the TOPgal reporter gene, a small number of TOPgal positive cells were found in the articular cartilage of adult mice. This finding suggests that Wnt signaling maintains a stem cell/progenitor cell bank in adult life to facilitate the regeneration and repair of articular cartilage .Among various factors leading to cartilage degeneration, abnormal mechanical stimulation plays a major role. Under physiological conditions, mechanomechanical signals from chondrocytes usually promote chondrocyte proliferation, enhance chondrocyte extracellular matrix (ECM) anabolism, inhibit inflammatory responses, and maintain cartilage integrity . For exaStudies on OA patients, surgery-induced OA mouse models, aged mice, and spontaneous guinea pig osteoarthritis model reveal that there is an upregulation of β-catenin expression along with the upregulation of MMPs and ECM degradation, which are indicative of chondrocyte hypertrophic differentiation . After m2+ pathways in a dose-dependent manner, and promotes chondrocyte proliferation and differentiation through the Wnt/β-catenin and Wnt/Ca2+ signaling pathways, respectively. These two pathways inhibit each other. After treating chondrocytes with the canonical Wnt signaling pathway inhibitor DKK1, they found that blocking the Wnt/β-catenin pathway would also lead to the dedifferentiation of articular chondrocytes by inhibiting the Wnt/Ca2+ pathway  mice could significantly reduce articular cartilage injury. Through inhibiting the progressive increase of joint matrix metalloproteinases (MMPs) activity in DMM animals . Also, bin vivo and in vitro models  through Wnt/β-catenin and Yap1 pathways. In contrast, activation of Piezo1/2 induced osteogenic differentiation by activating Wnt and YAP signaling pathway  are two key calcium channels that respond to mechanical forces, and recent studies have shown that the Wnt signaling pathway was involved in their mediated regulation of bone metabolism. Piezo1 channel responds to hydrostatic pressure (HP) and regulates Wnt expression (Wnt5b and wnt16) and ciliogenesis to promote odontoblast differentiation. Also, the Peizo1 agonist Yoda1 increased Wnt16 expression . The sti pathway . In addi pathway . The act pathway . HoweverIn conclusion, Wnt signaling plays an important role in regulating the fate and metabolism of cartilage chodrocytes in turns of mechanotransduction and inflammation. The impact of Wnt signaling on OA is intricate since loss and gain of function of the Wnt/β-catenin signaling pathway in chondrocytes both could potentially leading to joint damage or exacerbate osteoarthritis. While strong inhibition of active Wnt/β-catenin signaling in cartilage leads to complete cartilage destruction and cell death in 15-month-old mice with OA, loss of function of the Wnt inhibitor is prone to increase susceptibility to OA in both mice and humans .According to the Chinese Guidelines for the Diagnosis and Treatment of Osteoarthritis (2021 Edition), the current clinical treatment of OA is still based on relieving pain and inflammatory symptoms as well as joint replacement surgery, there is no effective treatment that can prevent or delay degenerative changes in the joint, and it is urgent to develop more targeted treatments that act on specific signal transduction pathway. At present, treatment of osteoarthritis remains largely limited to steroidal or nonsteroidal anti-inflammatory drugs that only relieve symptoms of pain and inflammation . With thDifferent inhibitors can act on different targets of Wnt signaling pathway and regulate anabolism and catabolism of chondrocytes, to achieve the purpose of improving OA . On the On the other hand, some inhibitors inhibit the transduction of Wnt signaling pathway by targeting pathway sites in the cytoplasm, or act to block the transcriptional activity of TCF/LEF in the nucleus. Intra-articular injection of GIN, a GSK-3 inhibitor, induced cartilage surface fibrillation and reduced glycosaminoglycan expression and chondrocyte dysfunction by downregulating the canonical Wnt signaling pathway . Li et aFurthermore, blockade of canonical Wnt signaling by two small molecule inhibitors, SAH-Bcl9 and StAx-35R, suppressed chondrocyte phenotypic transfer in preserved cartilage at early stages of OA, resulting in increased SOX9 and ACAN and decreased COL10A1 gene expression . DOT1L, In vitro, studies have shown that SM04690 can effectively inhibit the Wnt pathway, and Wnt gene expression is significantly downregulated 48 h after treatment of human MSCs. After 21 days, SM04690 induces downregulation of osteogenic gene expression and upregulation of cartilage gene expression  is the main therapeutic component. By evaluating treatment after anterior cruciate ligament transection combined with medial meniscectomy in rats, Xie et al. found that Achyranthes saponin targets Wnt3a, inhibits the Wnt signaling pathway, reduces inflammation and cartilage lesions, thereby playing a role in slowing OA . Huang eIn addition, dietary nutrient intake in daily life can also have an impact on the development of osteoarthritis. Several studies have now shown that some dietary micronutrient supplementation may protect against OA , which iIncreasing evidence suggests that Wnt signaling plays an important role in maintaining joint homeostasis, particularly in chondrocytes. When joints are subjected to abnormal mechanical loading, the balance of Wnt signaling in the body is disrupted, followed by cartilage damage, leading to osteoarthritis at last. Therefore, it is necessary to further understand the role of Wnt signaling pathway in chondrocyte homeostasis, and new targeted drugs or gene therapy targets can also be developed through this signaling pathway to provide a new direction for the diagnosis and treatment of mechanical load-mediated osteoarthritis and other diseases.When exposed to circulating hydrostatic pressure (HP), the expression levels of miR-27a/b, miR-146a/b, miR-140, and miR-365 and their target genes  were regulated in OA chondrocytes, by activating the Wnt/β-catenin pathway . IncreasThe first type includes miRs that directly act on Wnt signals, such as miR-154-5p, miR-92-3p, miR-497-5P, miR-26b, miR-410, miR-203 and miR-146a. Some miRs act on DKK-1, including miR-320a, miR-335-5p. Other miRs act on Frizzled, including miR-1 and miR-29 . Mao et In addition, it has been found that the Wnt/β-catenin pathway can significantly downregulate the downstream coding products MMP13, ADAMT5, IGFBP5, and HDAC4 by regulating specific miRs associated with OA, such as miR-27a/b, miR-140, miR-146a/b, and miR-365 .Secondly, there are miRs that act on β-catenin and related proteins, such as miR-29c-3p for Dsh/Dvl, miR-26b for GSK-3, and miR-142-3p for APC. And miR-320c and miR-10a directly acting on β-catenin. They can directly or indirectly act on β-catenin in the cytoplasm, regulate different processes such as cell proliferation, migration, and regeneration by transducing extracellular signals, and then affect the progression of OA . Hu et aFinally, some miRNAs contribute to the transcription of TCF/LEF in the chondrocyte nucleus. Studies have shown that miR-365, miR-222, miR-140 and others directly target HDAC that inhibits TCF-4 degradation. MiR-145, miR-101, and miR-615-3P target SOX-9 to affect the nuclear phosphorylation of β-catenin, and thus regulate the transduction of Wnt signaling pathway . Based oThere are two fates of chondrocytes, either undergoing endochondral ossification and being replaced by osteocytes, or becoming long-lasting articular chondrocytes residing on the joint surface. The Wnt signaling pathways play a critical role in both processes, especially in joint formation and articular cartilage homeostasis. Targeting the overactive Wnt/β-catenin signaling in OA process has emerged as a strong and promising preventive strategy against OA. Nevertheless, the modulators and networks responsible for the overactive Wnt signaling in OA are not well characterized. Although the modulators mentioned herein are not exhaustive, they can serve as clues to find new drug or gene therapies that target the Wnt signaling pathway for diagnosing and treating osteoarthritis and other degenerative joint diseases.


Heparan sulfate is a crucial extracellular matrix component that organizes structural features and functional protein processes. This occurs through the formation of protein–heparan sulfate assemblies around cell surfaces, which allow for the deliberate local and temporal control of cellular signaling. As such, heparin-mimicking drugs can directly affect these processes by competing with naturally occurring heparan sulfate and heparin chains that then disturb protein assemblies and decrease regulatory capacities. The high number of heparan-sulfate-binding proteins that are present in the extracellular matrix can cause obscure pathological effects that should be considered and examined in more detail, especially when developing novel mimetics for clinical use. The objective of this article is to investigate recent studies that present heparan-sulfate-mediated protein assemblies and the impact of heparin mimetics on the assembly and function of these protein complexes. Intercellular signaling and correct structural organization are largely mediated by the extracellular matrix, which functions to organize macromolecules in a local and temporal manner. This is necessary for multicellular life ,2, and aFor this purpose, heparin-based drugs including heparin-mimicking drugs are actively being developed ,13,14. HHeparan sulfate molecules are important components of multicellular organisms that allow for the correct structural and functional organization of the extracellular matrix. Heparan sulfates interact with various proteins through both electrostatic and hydrogen-bonding interactions . These aOne of the key ways in which heparan sulfates directly regulate cellular processes is by modulating the activity of growth factors. Growth factors are signaling molecules that are involved in cell proliferation, differentiation and migration. Heparan sulfates are able to interact with these molecules, promoting the formation of signaling complexes that activate downstream signaling pathways. This is evidenced by the complex formation of FGFR1 and its ligand, which stabilizes the formation of a protein–protein complex . StructuThe indirect functional role of heparan sulfate on cellular signaling brings up two possible sources for a wide range of physiological diseases related to uncontrolled signaling. The first is mutations in the direct binding interfaces of proteins that affect ligand binding and subsequent protein diffusion . The cruThe second potential cause of disease is irregularities in enzymes that release heparan-sulfate-associated proteins from their structural supports, resulting in free ligand diffusion across the extracellular matrix. Heparanase is an extracellular matrix enzyme that functions to cleave heparan sulfate chains, where abnormal expression and function has been shown to cause uncontrolled cell signaling and remodeling of the extracellular matrix ,52. It wIn general, heparan-sulfate-mediated protein complex formation plays a critical role in regulating various cellular processes, including cell proliferation, differentiation and migration, by modulating the activity and localization of signaling proteins. Accordingly, the binding and retaining of proteins by heparan sulfates in the extracellular matrix adds a level of complexity to intercellular signaling that is often unaccounted for. As such, the precise mechanisms by which heparan sulfates interact with different proteins to control cellular processes are still not fully understood. Still, our current knowledge of irregularities in the structural integrity of GAG-mediated protein assemblies caused by intrinsic or extrinsic forces emphasizes the significance of heparan sulfate as a structural component. Prospective studies on protein systems in the extracellular matrix should consider possible effects of GAG ligands and the formation of larger protein assemblies that might affect cellular signaling and matrix organization.Heparan sulfate is a crucial component for multicellular life, and it interacts with many different signaling proteins and systems. Although heparan sulfate is a very unspecific ligand due to its overall strong negative charge, various heparin mimetics have been developed that constitute different classes, from basic small saccharides to polysulfated oligosaccharides and non-carbohydrate mimetics ,57. GeneAs heparan sulfates take part in the structurally organization of the extracellular matrix, inflammatory and fibrotic events are highly dependent on the present heparan sulfate composition and function. This includes the careful control of heparan sulfate modeling by enzymes and subsequent formation of protein assemblies that regulate proinflammatory and anti-inflammatory cytokines . This baFurthermore, cancer progression is often associated with abnormal remodeling of the immediate extracellular matrix, which allows for elevated cell proliferation and differentiation based on compromised control mechanisms. Synthetic heparin mimetics have recently been produced that show antithrombotic and inhibitory activity of various enzymes such as Heparanase, P-selectin and the integrin VLA-4 to drastically reduce metastatic activity; the heparin mimetic efficiently blocked melanoma cell binding to endothelial cells under blood flowing conditions . This waBeyond the direct effects of heparin mimetics on enzymes and signaling proteins, changes that affect general protein–heparan sulfate interactions and protein assemblies can alter signaling behavior. An example of this is the antagonist removal from the immediate surroundings of a cell surface ,66, whicIn any case, the underlying disease mechanisms in relation to heparan sulfate or heparin dependence have to be examined in order for appropriate drug development to occur. Heparin mimetics can function as inhibitors against extracellular matrix deconstruction and remodeling  16], as, as16],