Curative therapy for sickle cell disease (SCD) currently requires gonadotoxic conditioning that can impair future fertility. Fertility outcomes after curative therapy are likely affected by pre-transplant ovarian reserve or semen analysis parameters that may already be abnormal from SCD-related damage or hydroxyurea treatment. Outcomes are also likely affected by the conditioning regimen. Conditioning with myeloablative busulfan and cyclophosphamide causes serious gonadotoxicity particularly among post-pubertal females. Reduced-intensity and non-myeloablative conditioning may be acutely less gonadotoxic, but more short and long-term fertility outcome data after these approaches is needed. Fertility preservation including oocyte/embryo, ovarian tissue, sperm, and experimental testicular tissue cryopreservation should be offered to patients with SCD pursing curative therapy. Regardless of HSCT outcome, longitudinal post-HSCT fertility care is required. Fertility is a long-standing concern for individuals with sickle cell disease (SCD) and their families. The risk of infertility with SCD curative therapy is a barrier to patient acceptance and provider referral for hematopoietic stem cell transplantation (HSCT) ,2,3. StaAuthors’ note on language: There are two linguistic assumptions in this manuscript. We note that biologic sex at birth is not synonymous with gender identity. In this review we use “girls and women” interchangeably with people with ovaries and “boys and men” interchangeably with people with testes. A second language concern is in the use of the word fertility. Fertility, the ability to conceive children, is not easily studied. In a clinical sense, fertility is defined by the absence of infertility. Infertility is defined by the failure to conceive after 12 months of unprotected heterosexual intercourse. When infertility is a concern, for any reason, measures of ovarian reserve and semen analysis are used to assess fertility and predict success of fertility preserving interventions and guide the intervention. In this paper, we use “fertility measures” as synonyms for ovarian reserve testing and semen analysis. Hormone values are also important in the evaluation of potential ovarian insufficiency or testicular failure causing infertility. An overview of relevant measures of gonadal function and their significance in male and female physiology is provided in Fertility Considerations in Girls and Women.Ovarian reserve is often assessed by measuring three values: Follicle Stimulating Hormone (FSH), Anti-Mullerian hormone (AMH), and antral follicle count (AFC). No single test is perfect, but the combination provides a good idea of ovarian function and egg supply.Follicle Stimulating Hormone (FSH): Ovarian reserve can be assessed by measuring FSH along with estradiol levels on menstrual cycle day 2–4. Normal basal levels are FSH < 10 IU/L and estradiol <60 pg/mL . A. A110]. SCD damages the testes which, like the ovaries, are exposed to inflammation and hypoxic-ischemic damage. This injury impairs normal spermatogenesis and causes testicular infarction ,116,117.n = 23) and previously treated with hydroxyurea during childhood (n = 15) found no significant differences in SA parameters between the two groups [n = 13), previously treated with hydroxyurea (n = 11), and currently receiving hydroxyurea (n = 6) and found no significant differences in testicular tissue [Limited evidence defines the effects of SCD treatments on male fertility. This literature primarily concerns the adverse effects of untreated SCD and hydroxyurea. Hydroxyurea treatment decreases sperm counts as demonstrated in a prospective study of 35 men with SCD . Six mono groups . Two stur tissue . In contr tissue . In the Other medications commonly used by people with SCD may also affect male fertility. Opioids and marijuana may disrupt the hypothalamic–pituitary–adrenal (HPA) axis and negatively affect male fertility . LimitedLimited post-cure fertility and pregnancy related data exist for men with SCD . Most reThe existing data mostly concerns outcomes after HSCT with myeloablative Bu/Cy regimens. Long-term follow-up of participants in the first multicenter center HSCT trial for SCD identified that among male participants, none had elevated LH/FSH levels, but 4/13 evaluated patients (31%) had low levels LH/FSH  and 10/13 (77%) had low testosterone levels . In cont2) [Some results of reduced intensity HSCT conditioning regimens are available. In a study comparing reduced dose Bu/Cy regimens to conventional doses , measure2) . Two men2) .Nonmyeloablative conditioning using alemtuzumab and 3 Gy TBI may be less gonadotoxic especially for boys/men given irradiation testicular shielding. Among the 72 men with SCD transplanted with this approach, 7 men reportedly fathered children , includiSperm cryopreservation: Sperm cryopreservation is used to preserve fertility in post-pubertal males. No studies specifically report on sperm banking procedures and subsequent reproductive outcomes among men with SCD. TMSC greater than 9 million optimizes pregnancy rates for intrauterine insemination (IUI), but pregnancy with IUI can be achieved with much lower TMSCs  [250,000) . For tho250,000) . Since m250,000) . Sperm e250,000) .Hydroxyurea’s negative effect on sperm count may impede sperm banking ,141. WheTesticular tissue cryopreservation: For prepubertal boys and patients unable to sperm bank, testicular tissue cryopreservation (TTC) is an experimental fertility preserving intervention currently offered at a limited number of centers. TTC requires unilateral testicular biopsy to obtain spermatogonial stem cells that are cryopreserved and may be used to produce mature sperm. Sperm maturation methods are under study with no successful pregnancies yet reported. Hydroxyurea may not damage spermatogonial stem cells, so may be continued, but in the absence of definitive data, this decision should involve shared decision making [n making . Males wn making ,133,143;n making .The TTC procedure involves testicular biopsy and sedation or general anesthesia. Typically, boys undergo the latter due to inability to tolerate sedation. The procedure takes under one hour. In the general population, testicular biopsy is low risk (2–3%) with minor procedural complications reported ,153,154.For boys/men, we suggest annual post-HSCT testing to include FSH, LH, and testosterone. Since men with normal hormone levels can still have impaired spermatogenesis, SA testing should also be done to evaluate gonadal function. Men with azoospermia early post-HSCT may not be infertile as spermatogenesis recovery can occur many years after HSCT . Men shoLess toxic curative approaches may make fertility preservation unnecessary in the future, but at present, fertility preservation is a fundamental component of SCD care. There are many uncertainties associated with counseling about fertility preservation for SCD. These include, but are not limited to, precise fertility risks with preparative regimens, pregnancy outcomes with preserved gametes, and spontaneous pregnancy rates. Incorporating this uncertainty into counseling is necessary since definitive data will take time to accrue. Given the potential for gonadotoxicity, even with non-myeloablative conditioning, fertility preservation is indicated for all patients with SCD pursuing curative therapy.The lack of universal access to fertility preservation before curative therapy is a medical, ethical, and practical concern. Many states have introduced bills to mandate insurance companies to cover fertility preservation procedures for patients who are facing potential infertility as a result of medical treatment (iatrogenic infertility). As of October 2021, eleven states mandate insurance coverage for fertility preservation. However, even states with fertility mandates limit coverage contingent on insurer and insurance type. Public and federal insurance do not cover fertility preservation except in Illinois where a public insurance program covers fertility preservation . Marked To provide indicated the pre- and post-HSCT fertility care to individuals with SCD, clinical care structures need immediate reform, hematologists and HSCT specialists need to engage fertility specialists as essential SCD care partners, and policy changes are needed to resolve unequal access to fertility care in SCD .


Recent observations suggest that autism spectrum disorder (ASD) co-occurs in people with a functional neurological disorder (FND), but little systematic data are available on the relationship between FND and autism. The study aimed to assess the self-reported autistic traits via a standardized questionnaire and the prevalence of previously diagnosed ASD among people with FND and their 1st-degree relatives. We performed a survey of members of the patient organization FNDHope, using a self-completed questionnaire for screening for autistic traits and ASD: the adult autism subthreshold spectrum (AdAS spectrum). There were 344 respondents diagnosed with FND with a mean age of 39.8 ± 11.6 years . Eight per cent of respondents volunteered a previous diagnosis of ASD, and 24% reported a 1st-degree relative with a formal diagnosis of ASD, mostly their children. We found that 69% of respondents had scores in the AdAS spectrum indicating a clinically significant ASD and 21% indicating autistic traits. Further studies are needed to provide more evidence regarding the prevalence of ASD in people with FND and how this may influence the aetiology, treatment selection and prognosis. Functional neurological disorder (FND) is a common neurological condition where the primary problem appears to be a genuine lack of voluntary access to movement and/or sensation, despite normal basic neurological function. People with FND most commonly present with abnormalities of movement control , sensory and cognitive disturbances or attacks that resemble epilepsy (dissociative seizures/non-epileptic attacks). This frequently causes disability and a poor quality of life , and mosThe aetiology of FND is not fully understood. Data suggest various risks, precipitant, and perpetuating factors. Previous stressful life events, including childhood or adult-life abuse, certain personality traits, anxiety and depressive disorders are significantly more common in patients with FND, compared to healthy and patient controls ,9. HowevFrequently, people with FND also present with functional somatic syndromes (FSSs), such as irritable bowel syndrome (IBS), fibromyalgia/widespread chronic pain (FM/CWP) or chronic fatigue syndrome (CFS) . DespiteSome emerging but inconclusive data show neurodevelopmental differences in people with FND and FSSs, especially autism spectrum disorder (ASD). In a cohort of 288 patients with FND, 9% were found to have co-existing neurodevelopmental conditions, with a higher frequency seen in those with dissociative seizures . AnotherUnderstanding if neurodevelopmental differences  are part of the pathophysiology of FND could lead to important insights into the aetiology and treatment. We hypothesized that autistic traits would be common among people with FND and assessed this using a validated self-completed questionnaire. We also studied the prevalence among people with FND and in their 1st-degree relatives of previous neurodevelopmental conditions with a special focus on ASD.In this cross-sectional observational study, we administered an online survey to subjects who reported a diagnosis of FND received from a medical professional.Recruitment was achieved by disseminating the questionnaire through the patient support groups FND Hope UK and the FND Hope International website and social media to individuals who had consented to be contacted with information of this sort. In total, these accounts have more than 7200 followers. The research protocol included some questions for gathering the sociodemographic data, and the invitation included information that neurodevelopmental differences, including ASD and their possible relationship to FND, were the topic of the study. Autistic traits were assessed by an online English version of the adult autism subthreshold spectrum (AdAS Spectrum) self-reported instrument developed and validated by Dell’Osso et al. .The AdAS spectrum has 160 dichotomous items (yes/no). It is organized into seven domains: childhood/adolescence 21 items), verbal communication (18 items), non-verbal communication (28 items), empathy (12 items), inflexibility and adherence to routine (43 items), restrictive interests and rumination (21 items); hyper-hypo reactivity to sensory input (17 items). Its design best captures the new DSM-5 criteria referring, not only to the core manifestations of ASD, but also to the attenuated and atypical symptoms, the personality traits, and the behavioral manifestations that may be present in partial forms . Further items, vA version of the AdAS spectrum was created via the Microsoft form platform and shared open access over a period of 6 weeks from 15 November to 27 December 2021. The data were returned in an anonymized fashion without any personal identifiers. The respondents were also asked about basic demographics, a previous medical professional diagnosis of neurodevelopmental conditions, such as autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning disabilities (LDs), intellectual disability (ID), and diagnosis of these conditions in 1st-degree relatives.t-test, Kruskal–Wallis and Mann–Whitney tests for the continuous variables, as required. The results were considered statistically significant at a 2-tailed p < 0.05. A post-hoc Bonferroni correction was applied for multiple comparisons, when required. The statistical analysis was performed by IBM SPSS Statistics 26.As a preliminary step, the cases with high levels of missing data in the AdAS spectrum (>10% in the same domain or the whole questionnaire) were removed. To account for the non-responded items, the total AdAS spectrum score and domain scores were weighted for the number of valid answers. The weighted score was calculated as the sum score multiplied by 160 (number of items in the questionnaire) and divided by the number of completed items. Item responses were preliminarily tested for asymmetry and kurtosis. Age was calculated as the year of birth to the survey year (2021). Descriptive statistics  were used to summarize the data. Averages are presented as mean ± standard deviation (SD). Group comparisons were performed using xi2 for the categorical variables and the The FND Hope UK and FND Hope International social media have approximately 7200 followers. The total number of people who saw the post is unknown, however, 361 people submitted responses. The study was open for six weeks, with 80% of the responses gathered during the first week of publication. This suggests the study was less visible over the following weeks when several invitations to the studies from other groups were published on subsequent days.Of the total participants, 359 consented for their answers to be analyzed. Fourteen participants were directly excluded as they provided incomplete demographic data or had a significant number of responses missing from the questionnaire. One participant was excluded due to not having a formal diagnosis of FND. A total of 344 participants with FND were analyzed.n = 310, 90.1%, p < 0.001) with female gender identity  and had a mean age of 39.8 ± 11.6 years. A previous diagnosis of ASD was present in 27 (7.8%), ADHD in 23 (6.7%), and LD/ID were present in 23 (6.7%). Of the 143 (41.5%) participants that declared having a 1st-degree relative with a neurodevelopmental disorder, ASD was significantly more common (24%), followed by ADHD (11.6%) and LD/ID (5.8%), p < 0.001. Of those declaring having a first-degree relative with ASD, this was their children in 69.9%, p < 0.001.The data are presented in n = 91, 26.5%), we asked whether the other parent was also diagnosed. This question was answered positively by 13 (14.2%), mostly accounted for ADHD  and less frequently for ASD  or learning disabilities .Among those who indicated that they had children with a neurodevelopmental condition (p < 0.001). Scores in the AdAS spectrum indicating a clinically significant ASD was related to the previous diagnosis of neurodevelopmental disorders (p = 0.003). Accordingly, the questionnaire classified all participants who reported a previous diagnosis of ASD, among the ASD spectrum . However, higher scores in the AdAS spectrum were not related to the diagnosis of neurodevelopmental disorders in first-degree relatives. All 12 respondents with different gender identities from female/male were classified in group C .The AdAS spectrum questionnaire data are summarized in p = 0.005) and group A (p = 0.006). There were no significant age differences between groups A and B (p = 0.4).Group C showed a mean age significantly lower, when compared with both group B  score between a chronic fatigue syndrome group and a healthy control group, and both scored lower on the AQ than the group with ASD . StudiesWe used the AdAS spectrum to assess the lifetime autistic traits . AlthougThe threshold values were established in a study including three groups: ASD, subjects with autistic traits, and healthy controls. Trained psychiatrists made the diagnosis, according to DSM-5, and the subjects self-completed the AdAS spectrum and the AQ. The best threshold value for identifying ASD was a score of 70, which showed a good agreement with the diagnosis performed, according to the DSM-5 criteria (specificity of 0.910 and sensibility of 0.923) and 43 for the autistic traits (specificity of 0.833 and sensitivity of 0.849) . In the n = 58, 16.9%). The population variation in ASD occurrence is strongly related to genetic influences, with an estimated heritability of 80% [Approximately 25% of respondents reported having a 1st degree relative with ASD, mainly their children (y of 80% . Given ty of 80% ,52,53, bObsessionality is a common feature among people with ASD . TherefoWhy individuals with autism may have an increased vulnerability to developing FND, is still an unanswered question. Characteristics of the brain structure, function and connectivity may play a role, but there may also be a higher exposure to known risk factors for FND ,9. ExposThere are key links between the pathophysiology of ASD and FND in attentional processing, sense of agency and interoceptive processing. While the way in which these interact in both ASD and FND is still incompletely understood, the overlap of these features is intriguing. A key piece of missing understanding is why, despite some core underlying pathophysiological similarities, only a proportion of people with ASD develop FND and why this occurs typically in the 3rd to 4th decade, despite the lifelong nature of ASD.We acknowledge a number of limitations to our study. Almost 70% of people with FND in our sample scored in the range suggesting autism spectrum. Although we have anecdotally recognized several individuals in our clinics with probable ASD, this extremely high prevalence was surprising to us and makes it important to consider potential sources of bias in our data. We performed an online survey of a community with over 7200 members and gained only 361 respondents. Specific data on ethnicity, socioeconomic status and educational attainment were not recorded. There are, therefore, several possible biases here. First, the people who are part of the FND Hope online community may not be fully representative of all people with FND. Second, there may well have been a response bias for those with a specific interest in neurodevelopmental disorders or ASD. Online surveys, such as this, tend to favour younger patients and those with computer skills and may fail to reach those with poorer educational levels or more social deprivation. Although we chose a validated instrument to measure the autistic traits and ASD, this is not a substitute for a formal in-person interview and multidisciplinary assessment, and we cannot exclude that participants misunderstood or otherwise failed to give an accurate answer to the questions.Our results revealed a high prevalence of people with FND who scored above the threshold for autistic traits and ASD on a validated self-completed questionnaire and who have a previous diagnosis of a neurodevelopmental disorder or 1st-degree relatives with neurodevelopmental disorders.Further research is needed to understand the intersection between autism and FND. This is particularly important when approaching the FND diagnosis and delivering treatment, as people with autistic traits and ASD may have specific needs that require to be identified and properly integrated into treatment.


HTLV-1 uveitis (HU) is the third clinical entity to be designated as an HTLV-1-associated disease. Although HU is considered to be the second-most frequent HTLV-1-associated disease in Japan, information on HU is limited compared to that on adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Recent studies have addressed several long-standing uncertainties about HU. HTLV-1-related diseases are known to be caused mainly through vertical transmission (mother-to-child transmission), but emerging HTLV-1 infection by horizontal transmission  has become a major problem in metropolitan areas, such as Tokyo, Japan. Investigation in Tokyo showed that horizontal transmission of HTLV-1 was responsible for HU with severe and persistent ocular inflammation. The development of ATL and HAM is known to be related to a high provirus load and hence involves a long latency period. On the other hand, factors contributing to the development of HU are poorly understood. Recent investigations revealed that severe HU occurs against a background of Graves’ disease despite a low provirus load and short latency period. This review highlights the recent knowledge on HU and provides an update on the topic of HU in consideration of a recent nationwide survey. Human T-lymphotropic virus type 1 (HTLV-1) was the first retrovirus identified to cause human diseases . The focSince the discovery of HTLV-1, several clinical entities of HTLV-1-associated disease have been identified, such as adult T-cell leukemia/lymphoma (ATL) ,6,7, HTLThe characteristics of HU have been determined by various clinical and basic research studies three decades ago ,18,19, aAlthough three decades have passed since the initial awareness of HU, several uncertainties related to this condition remain. Recently, several notable findings addressing these uncertainties were reported. This review highlights the latest information on HU and provides an update on the topic of HU in consideration of a recent nationwide survey in Japan.HU is defined as intraocular inflammation caused by HTLV-1 infection , in whicVitreous opacity is the most frequent ocular feature and is responsible for foggy vision, floaters, blurring of vision, and vision loss. Retinal vasculitis, identified as vascular leakage on fluorescence angiography, is also seen frequently, and it affects retinal function, resulting in impaired vision. Infiltrating cells, including HTLV-1-infected cells and inflammatory cells, which damage intraocular tissues, are seen in the anterior chamber and vitreous. Recurrence of HU is seen in around half of HU patients, leading to irreversible vision loss ,31,32.The current therapy for HU includes steroids through topical, sub-Tenon, and systemic administration. For focal treatment, topical steroid eye drops are used initially, followed by sub-Tenon injection of the steroid in unresponsive cases. However, systemic treatment  is needed in more than 30% of HU patients .The reported ocular complications of HU include glaucoma, cataract, cystoid macular edema, and keratoconjunctivitis sicca . In termJapan has approximately 1 million HTLV-1-infected individuals and is considered the most HTLV-1 endemic country among developed nations . In the Our latest nationwide survey of ophthalmic care for HTLV-1 in Japan provided new information and identified several concerns related to HU . The surThis high percentage of testing for HTLV-1 antibodies in ophthalmology is unique to Japan. In the three decades since the establishment of the entity of HU, the majority of facilities in Japan started performing tests for HTLV-1 antibodies when evaluating the differential diagnoses of uveitis. This implies that ophthalmologists in Japan are well aware of HTLV-1 infection .In Japan, HTLV-1 antibody screening of all donated blood has been conducted by the Japanese Red Cross since the latter half of the 1980s. Since then, the implementation of infection control measures has contributed to eliminating HTLV-1 infections. Acknowledging the importance of HTLV-1 infection, the Japanese government also started routine antenatal HTLV-1 antibody screening of pregnant women in 2011 as one of the priority measures of “Anti-HTLV-1 Initiatives by the Japanese Government” ,39. ThesThe lack of a measurable decrease in the number of new patients with HU in our nationwide survey could be explained by referring to a recent study by the Japanese Red Cross Blood Center ,40. A JaThis is contrary to the transmission route for the other HTLV-1-associated diseases, ATL and infective dermatitis, which are believed to mainly occur through a vertical transmission route ,42. So fConsidering the lack of a decline in the incidence of HU in our nationwide survey, despite attempts to eliminate the transmission route described above by the Japanese Red Cross and government, we suspected that there must be a horizontal transmission route for the development of HU and started investigating the transmission route for HU patients and investigating their long-term clinical course.By analyzing the HTLV-1 infectious status of HU patients’ families and the clinical manifestations of HU patients during medical care, we determined that a horizontal transmission route was responsible for HU. In addition, we observed that horizontally transmitted HU caused severe and persistent ocular inflammation during follow-up  43]..43].In the current environment of the non-declining trend of HTLV-1 infections, the existence of horizontal transmission of HU should be acknowledged because it can result in more severe inflammation than vertical transmission. Thus, physicians should take the route of infection into consideration when providing medical care to patients with HU.Clinically, the proviral load  is one of the most important factors in the development of HTLV-1-associated diseases . In termThrough virologic analysis of HU patients, along with systemic investigations, we found that severe HU occurred in young HTLV-1 carriers after the onset of Graves’ disease and subsequent administration of methimazole, even though the provirus load was low  Figure .This phenomenon implies that the acceleration of the development of HU occurs in HTLV-1-infected patients who develop Graves’ disease and receive methimazole treatment despite a low provirus load and short latency period. This observation suggests the need for physicians to be cautious when Graves’ disease patients complain of blurry vision and that physicians, including endocrinologists and ophthalmologists, should rule out HTLV-1 infection in the differential diagnoses in such patients.A possible mechanism for this association between HU and Grave’s disease is that thyroid hormones and methimazole might stimulate a small number of circulating HTLV-1-infected cells, which might contribute to the breakdown of the blood–ocular barrier, resulting in severe ocular inflammation. However, further analysis is needed to clarify this phenomenon.Biologics, such as tumor necrosis factor (TNF)-α antibody, were introduced in the late 1990s as a molecularly targeted therapy for inflammation ,50,51,52It is estimated that there is a large number of patients with inflammatory diseases, such as rheumatoid arthritis, who need to receive biologics despite having HTLV-1 infection . HoweverIn the field of ophthalmology, no evidence has been found for prescribing biologics to HTLV-1 carriers. In fact, our nationwide survey determined that the measurement of HTLV-1 antibodies is not performed in 50.0% of facilities prescribing biologics, and the reason commonly stated for this was a lack of knowledge regarding whether or not the measurement should be performed .In response to this survey, we started investigating the effects of biologics on the eye under HTLV-1 infectious conditions in vitro. At first, we focused on infliximab, the first biologic for inflammatory diseases , and fouAlthough these reports provided new evidence regarding the ocular safety of the use of biologics for inflammatory diseases in HTLV-1 carriers, which would be clinically useful for ophthalmologists, further analysis, including evaluation of other biologics, is needed in the future.Although HU is the most well-known and most studied ocular manifestation of HTLV-1, the other HTLV-1-associated disease entities might also present ocular symptoms. Several surveys that enrolled a large number of patients found that HAM sometimes occurs along with HU ,26,36. HThe most frequently seen manifestation is intraocular infiltration of ATL cells. We recently found that infiltration of ATL cells in the eye produces a specific sign, which we termed the “knob-like ATL cell multiple ocular infiltration” (KAMOI) sign, from the ocular surface to the retina ,62,63 F. This waThe second-most frequent ocular manifestation in ATL patients is CMV retinitis  Table 3Table 3. Recently, a new ocular finding was reported in the eye during the course of ATL. ATL is a life-threatening hematological disease with a poor prognosis that was previously reported to have a poor survival rate despite chemotherapy . RecentlRecently, several long-standing unresolved issues related to HU, such as its transmission route and factors associated with its development, have been clarified. Frequent and new ocular manifestations of HTLV-1-associated ATL have also been identified. In addition, a nationwide survey raised new issues about HU and addressed several concerns, such as the safety of biologics in terms of the eye in the presence of HTLV-1 infections. However, information on HU is still insufficient compared to that on ATL and HAM. Further clinical and basic investigations of HU are needed to prevent vision loss among HTLV-1-infected individuals.


Trigeminal neuralgia is a recurrent unilateral transient electroshock-like pain. Fu’s subcutaneous needling (FSN), used to treat the musculoskeletal problems, has not been reported in this field.The pain extent of case 1 had no reduction after the previous microvascular decompression, the pain of case 2 relapsed 4 years after the microvascular decompression.Postsurgical trigeminal neuralgia.FSN therapy was applied on the muscles around the neck and face area, which the myofascial trigger points were palpated in these muscles. The FSN needle was inserted into the subcutaneous layer and the needle tip was pointed toward the myofascial trigger point.The following outcome measurements were observed before and after treatment, including numerical rating scale, Barrow Neurology Institute Pain Scale scores, Constant Face Pain Questionnaire scores, Brief Pain Inventory-Facial scores, Patient Global Impression of Change scores, and medication dosage. The follow-up surveys were made after 2 and 4 months respectively. The pain of Case 1 was significantly reduced after 7 times FSN treatments and the pain of Case 2 was even disappeared after 6 times FSN treatments.This case report suggested that FSN can relieve postsurgical trigeminal neuralgia safely and effectively. Clinical randomized controlled studies are needed to be further conducted. The International Classification of Headache Disorders and International Classification of Orofacial Pain defined TN as “a recurrent unilateral transient electroshock-like pain that has a sudden onset and termination, limited to the distribution of 1 or more divisions of the trigeminal nerve and triggered by harmless stimuli.” Touching the face, talking, eating, or drinking can all cause fierce pain. The nature and severity of the pain maybe related to delayed diagnosis, fear of pain, side effects of medications, and lack of psychological support. European studies based on epidemiology and genetics have found that the lifetime prevalence of TN is 0.16% to 0.3%, and the incidence rate per 100,000 people a year is 12.6 to 27.0. TN affects women (60%) more than men (40%). TN patients have increased symptoms such as anxiety, depression, insomnia, affecting basic psychological, physical, social needs and activities of patients, and TN is even called suicide disease.,4 Microvascular decompression (MVD) is the first-line surgical therapy for medically refractory patients. Lars Bendtsen et al reported that MVD had a good effect, and 62% to 89% of patients exhibited no pain at follow-up (3–11 years later), but Giorgio Cruccu hold different opinions, he reported about 50% of TN patients also have persistent pain in the same area in addition to the characteristic paroxysmal episodes.Trigeminal neuralgia (TN) is a disturbing chronic pain condition on 1 side of the face.  which are hard, discrete, and palpable nodules in a taut band of skeletal muscle. FSN therapy is often performed by the reperfusion approach, the process of contracting a muscle for seconds, combined with the swaying movement. FSN therapy has a reliable effect in reducing pain, especially musculoskeletal problems.–12 However, so far, there is no report of FSN for TN. In this case report, we presented 2 cases of TN patients who have persist pain after the surgery of MVD. The symptoms were surprising significant relieved or eliminated after several sessions of FSN treatments.Fu’s subcutaneous needling (FSN) therapy is a new type of acupuncture therapy developed in 1996. With disposable FSN needles, FSN mainly stimulates the subcutaneous layer adjacent to myofascial trigger points (MTrPs),Two TN patients who received MVD surgery previously were treated by FSN therapy in Yangzhou TCM Hospital, East China, from October 2020 to April 2021. The authors obtained the written consents of the patients to describe their illness and willingness to publish their case reports. We did not use patient data that could allow their identification. The patients lied in a supine position. We palpated the MTrPs with the relevant muscles around the neck and face area, and then marked them. After locating the MTrPs, chose the insertion points around the MTrP as the guidance of FSN practice. Each insertion point was disinfected with iodophor. The FSN needle On October 24, 2020, a 52-year-old woman visited our clinic. She received a hyperthyroidism operation 20 years ago, had anemia for 20 years. After receiving 2 MVD surgeries  for her TN problems, her symptoms remained the same till then. The details about her medical history showed as Table The NRS was 8 during rest time, and reached to 10 while opening mouth, chewing, talking, and blowing at the first visit. The local facial muscles were bruised due to longtime massage by herself. The patient felt soreness during palpation of the sternocleidomastoid muscle on the left side of the neck and the left buccal muscle. A lot of active MTrPs were found in the left biceps, trapezius, scalene, sternocleidomastoid, and buccal muscles while physical examination of her neck and upper back.Seven sessions of FSN therapies were performed within 2 months. After treatments, her condition was significantly improved, and pain only occurred while in cold weather or in the fatigue state Fig. . The evaA 46-year-old female with a history of Hashimoto thyroiditis under medicine for maintaining normal thyroid function and mild anemia under the medication of iron supplements orally was treated in our clinic. The details medical history is listed in Table  Previous studies have reported the effectiveness of FSN in the treatment of neck pain, low back pain,–22 Lateral epicondylalgia and other painful diseases. The targets of treatment in the previous mentioned problems are MTrPs. In this article, the 2 cases are classified as typical TN according to the diagnostic classification. Myofascial trigger point (MTrP) is a major cause of muscle pain, characterized with a hyperirritable spot due to accumulation of sensitized nociceptors in skeletal muscle fibers. As shown in Figure  The transverse cervical nerve, the skin below the chin area, that is, the transverse cervical nerve drawn in yellow on the front of the neck in Figure  The distribution of referred pain pattern and trigeminal nerve was almost similar, so our participants had treated the TN with FSN, and the results were unexpectedly good.TN is currently 1 of the neuropathic diseases that are difficult to treat. The pain is very severe that patients lose their confidence in life. Although many treatment methods are available , and the effect is not satisfactory. Some can only temporarily relieve the pain, and others have many sequelae. Although the conditions of many patients initially improved during first-line treatment, most treatments tend to lose efficacy over time, so new treatment options are necessary. have evaluated the end-plate noise recorded using the skeletal muscle myofascial trigger spot (MTrS) in rabbits to determine the electrophysiological effect of FSN. The evaluation of rabbit skeletal muscle MTrS by recording end-plate noise, which is equivalent to MTrP in human muscle, confirmed the effects and possible pathways of FSN on electrophysiological phenomena. After the ipsilateral distal FSN treatment, MTrS irritation seems to be suppressed, the proximal FSN treatment has a better effect than the distal treatment. Based on this study, and the combined proximal and distal treatment were performed in the 2 cases. The cases in this paper show that FSN also has a good effect on head and neck MTrPs, but there is no comparison between the proximal and distal treatment efficacy, which will be observed in the future. Therefore, in addition to examining the patient facial muscles, the head, neck, shoulders, upper limbs, and thoracodorsal muscles were examined for MTrPs. Case 2 is more complicated than Case 1. In Case 2, the MTrPs were detected in the head and neck splenius, galea aponeurotica, and serratus anterior muscles.For the mechanism research of FSN therapy, Fu ZH et al found that fascia tissue is related to the etiology of capsular neuropathy, emphasizing the importance of general connective tissues (except ligaments) in peripheral nerve compression. Although the pathogenesis of TN is still not fully understood, neurovascular conflict is still the most accepted theory at present. Many factors can make arteries narrow, such as arteritis, and arterial embolism, but the muscle with MTrPs is the most common factor that changes the diameter of arteries. The treatment mechanism of FSN is speculated to be similar to that of the above methods. FSN treatment aims to restore and enhance muscle function by eliminating MTrPs, alleviating muscle tightness and cramps, and reducing pain.,7 These 2 cases of TN in this paper had left and right facial pain, and local tissue nutrition or energy supply was not available. When the action lasted for a period of time, the local blood supply was slightly relieved. When the muscles of the neck and facial on the ipsilateral side were too tight , they affected the blood supply in the head and face.Antonio et alThe advantage of FSN therapy is to propose a targeted treatment plan on the basis of the individual differences of each patient. According to the anatomy and treatment experience, the MTrPs can be found on the ipsilateral head, face, neck, shoulders, and chest . FSN therapy can greatly improve local microcirculation through swaying movement and reperfusion approaches, enhancing the nutrient supply and blood flow of the distribution of the trigeminal nerve and the surrounding soft tissues, improving or eliminating local inflammation and edema, and realizing a significant relief in symptoms.The cases in this report had limitations as follows. First, there was only a few treatment cases were observed. Secondary, there was no more accurate laboratory examination, like MRI to be conducted to determine the changes in the brain imaging after FSN treatment of these 2 cases. Third, we could not provide a perfect conclusion of the possible mechanism for the FSN treatment to TN, further randomized clinical trial for mechanism discussion was required for further research.FSN therapy used to be clinically effective for musculoskeletal pain, with definite curative effect and low side effects. This case report presented that FSN significantly relieved or even completely eliminated the refractory pain of TN undergone MVD. This report provides some clinical evidence regarding the efficacy of FSN on nonmusculoskeletal pain. However, the detailed mechanism of FSN on TN needs to be studied in the future.We are grateful to the patients for their participation in the study and allowing for publication of this case report.Conceptualization: Youling Gao, Jian Sun.Investigation: Youling Gao.Methodology: Zhonghua Fu, Li-Wei Chou.Project administration: Youling Gao, Jian Sun.Supervision: Jian Sun, Zhonghua Fu.Validation: Youling Gao, Jian Sun.Visualization: Youling Gao, Zhonghua Fu.Writing – original draft: Youling Gao, Po-En Chiu.Writing – review & editing: Zhonghua Fu, Li-Wei Chou.


WNT2B and WNT5A mRNA levels were upregulated after Jagged1 treatment. In contrast, the Wnt inhibitors DKK1, DKK2, and SOST mRNA levels were downregulated. Recombinant WNT5A, but not WNT2B, significantly promoted in vitro mineral deposition by hDPSCs. Wnt signaling inhibition using IWP-2, but not DKK1, inhibited Jagged1-induced alkaline phosphatase (ALP) activity, mineralization, and osteo/odontogenic marker gene expression in hDPSCs. In conclusion, Jagged1 promoted hDPSC osteo/odontogenic differentiation by modulating the non-canonical Wnt pathway.Osteoblast differentiation requires the interaction of various cell signaling pathways to modulate cell responses. Notch and Wnt signaling are among the crucial pathways that control numerous biological processes, including osteo/odontogenic differentiation. The aim of the present study was to examine the involvement of Wnt signaling in the Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells (hDPSCs). The Wnt-related gene expression was analyzed from publicly available data of Jagged1-treated human dental pulp cells. The mRNA expression of Wnt ligands  and Wnt inhibitors  were confirmed using real-time polymerase chain reaction. Among the Wnt ligands,  Wnt signal cascades are divided into the canonical and non-canonical pathways. Canonical Wnt signal transduction occurs via β-catenin accumulation in the cytosol that subsequently translocates into the nucleus, modulating target gene expression2. The non-canonical Wnt pathway comprises the calcium-dependent pathway and planar cell polarity cascades, which do not require β-catenin accumulation3. Wnt/β‐catenin signaling activation enhanced the osteogenic differentiation of human dental pulp stem cell (hDPSCs)4, apical papilla stem cells5, and human periodontal ligament stem cells (hPDLSCs)8. These findings suggested that Wnt signaling was involved in the osteogenic differentiation of dental-related cells.The Wnt pathway has crucial functions in various processes underlying the development and maintenance of tissue homeostasis9. Direct binding between a Notch ligand and receptor leads to receptor cleavage, releasing the Notch intracellular domain (NICD). This domain then translocates into the nucleus, forms a complex with other transcription factors, and activates Notch target gene transcription10. Previous studies demonstrated that a Notch ligand, Jagged1, potentiated osteo/odontogenic differentiation in various cells isolated from dental tissues, including human dental pulp cells, stem cells isolated from human exfoliated deciduous teeth, and hPDLSCs12. Therefore, Notch signaling has been demonstrated to play an important role in osteo/odontogenic differentiation.Notch signaling is involved in many cell responses during development and homeostasis15. Glycogen synthase kinase 3 beta (GSK-3β), a component of the Wnt/β-catenin signaling cascade, is involved in the temporal switch between Notch and Wnt signaling in mouse myogenesis13. GSK-3β modulated Notch signaling by directly phosphorylating NICD14. NICD activity stimulated the effect of Lymphoid enhancer-binding factor 1 (LEF‐1), a transcription factor involved in the canonical Wnt/β-catenin signaling, specific promoters15. However, the crosstalk that occurs between these two pathways to regulate hDPSC osteogenic differentiation remains unresolved. The aim of the present study was to investigate the participation of Wnt signaling in Jagged-induced osteo/odontogenic differentiation in hDPSCs.Cross-regulation between the Notch and Wnt pathways through their components has been reported in previous studiesThe isolated cells exhibited a spindle-shaped fibroblast-like morphology. Surface protein analysis revealed the expression of mesenchymal stem cell (MSC)-related surface markers (CD44 and CD105) and the absence of the hematopoietic cell marker CD45   immobilized surfaces were used as the control. Increased ALP staining and mineralization were observed at all time points in the Jagged1-treated group  were selected to validate the RNA sequencing results using real-time quantitative polymerase chain reaction. Among the Wnt proteins, WNT2B and WNT5A mRNA levels were upregulated, whereas WNT5B and WNT16 were downregulated after Jagged1 treatment. Furthermore, the canonical Wnt inhibitors DKK1, DKK2, and SOST exhibited decreased mRNA levels in the Jagged1-treated hDPSCs  ; however, ALP and COL1A1 mRNA levels were decreased compared with the control  /bone morphogenetic proteins (BMPs), Notch, Hedgehog, neural epidermal growth factor-like 1 protein (NELL-1), and Wnt/β-catenin, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and extracellular regulated-signal kinase (ERK) signaling, induce osteogenic lineage commitment and differentiation of MSCs19. All these pathways act in concert to regulate the osteo/odontogenic differentiation processes and ultimately bone formation. Among these key signaling pathways, Notch and Wnt signaling are highly conserved pathways that control several developmental processes, including bone formation and regeneration21. Notch exhibits inhibitory effects on osteogenic differentiation at the initial commitment stage, whereas it promotes terminal differentiation. The Wnt pathway modulates osteogenic differentiation via an interaction with the Runx2 promoter18. The previous publications by our group and other groups discussed the multi-communication crosstalk between Notch and other signaling pathways during osteogenic differentiation. Understanding the integration between signaling pathways provides insight into the molecular mechanisms that govern stem cell differentiation. The present study investigated the potential crosstalk between Notch and Wnt signaling in regulating hDPSC osteo/odontogenic differentiation. The isolated cells exhibited the characteristics that met the criteria for mesenchymal stem cells22. However, it should be noted that these cells were heterogeneous in nature. The responses of specific subpopulations require further analysis.Osteoblast differentiation requires orchestrated crosstalk between cell signaling pathwaysOur results revealed that Jagged1-mediated Notch activation upregulated several Wnt-related genes, including the canonical and non-canonical Wnt ligands WNT2B and WNT5A, respectively. Furthermore, WNT5A treatment promoted mineralization, suggesting that Wnt5A might participate in the Notch-induced osteo/odontogenic differentiation of hDPSCs. The inhibition experiments revealed that IWP-2, but not DKK1, attenuated the osteo/odontogenic differentiation of Jagged1-treated hDPSCs. These results imply the involvement of the non-canonical Wnt, WNT5A, in the Jagged1-induced mineralization in hDPSCs.25. Protein kinase C delta (PKCδ) was previously reported to interact and phosphorylate the NICD, leading to NICD stability and increased nuclear translocation. Jagged1 treatment increased the binding between NICD and PKCδ, thereby promoting Notch activation23 In addition, Jagged1 attenuated transcription factor TWIST expression, which is a negative regulator of osteogenic differentiation25. Several Notch components were expressed in the tissue adjacent to the pulp capping area, which influenced the differentiation of pulp cells into odontoblast-like cells in response to pulp injury26. Therefore, the activation of Notch signaling at the specific sites is crucial to control odonto/osteogenic differentiation. These results demonstrated that Notch positively regulates osteoblast differentiation. Similarly, our results indicated that Jagged1-mediated Notch activation drove hDPSCs toward the osteo/odontogenic lineage as demonstrated by increased ALP staining and mineral deposition. However, the effect of Notch signaling on osteoblast differentiation remains unresolved. Notch signaling attenuated osteoblast differentiation in murine bone marrow stromal cells by repressing the transactivating ability of Runx2 through Hes/Hey protein activity27. Thus, the effect of Notch signaling on osteogenic differentiation depends on the cell type and is stage specific28.Among the Notch ligands, the effectiveness of Jagged1 on Notch activation and osteo/odontogenic induction was demonstrated in previous studies29. Therefore, we hypothesized that this non-canonical Wnt might be involved in Notch-induced osteo/odontogenic differentiation. Similarly, WNT5A was previously reported to promote osteogenic differentiation of several cell types e.g., human bone marrow-derived mesenchymal stem cells30 and human adipose tissue-derived mesenchymal stromal cells31. Further, functionalized biomaterial-mediated activation of WNT5A enhanced the osteogenic lineage commitment of hMSCs in vitro and rat calvarial defect healing32. These results indicate the important role of WNT5A in osteogenesis. However, previous reports observed that WNT5A exerted an inhibitory effect on osteoblastic differentiation of an immortalized human periodontal ligament fibroblast cell line33. Similarly, WNT5A gene silencing promoted mineralization in human osteoblasts obtained from osteoarthritis patients34. We speculated that the different cell types and inflammatory condition might alter cell responses and lead to inconsistent findings.In the present study, Wnt signaling-related genes were differentially expressed in hDPSCs after Jagged1-treatment. WNT2B and WNT5A were among the most highly upregulated genes. We observed that WNT5A promoted mineral deposition in hDPSCs, Additionally, the osteo/odontogenic -related genes were upregulated concomitantly with the downregulation of ALP during the mineralization stage of osteo/odontogenic differentiation35. In addition, we utilized IWP-2, which attenuates Wnt protein transportation and secretion. Hence, both canonical and non-canonical pathways are inhibited36. We found that the effect of Jagged1 was inhibited only when the cells were treated with IWP-2, but not DKK1, as demonstrated by the decreased mineral deposition along with downregulated osteo/odontogenic -related genes. In addition, the β-catenin accumulation and Axin2 mRNA expression in Jagged1-treated hDPSCs had a similar profile compared with control, indicating that canonical Wnt signaling was not activated during Notch activation  were pivotal for HSC proliferation and maintenance41. Notch signaling activation downregulated the expression of Wnt inhibitors (Sost and Dkk), thereby promoting bone formation by osteocytes41. Notch and canonical Wnt activation were demonstrated to promote bone regeneration and healing in a mouse cortical defect model42. In contrast, negative regulation between these signaling pathways has also been reported. Canonical Wnt promoted MSC differentiation towards the osteoblast lineage; however, Hes1-induced Notch activation attenuated this effect of canonical Wnt, thereby inhibiting MSC osteogenic differentiation43. Crosstalk between Notch and canonical Wnt was observed during the early stages of intramembranous bone regeneration44. Notch regulated osteoprogenitor cell proliferation, and this effect was terminated by canonical Wnt activation, leading to osteogenic differentiation44. Similarly, the NICD was reported to occupy and inhibit T-cell factor/lymphoid enhancer factor at its binding functional domain, suggesting the underlying mechanism by which Notch exerts an inhibitory effect on the canonical Wnt signaling pathway46.Crosstalk between Notch and Wnt signaling was also reported during bone formation and regeneration47. However, further investigation is required to prove this hypothesis. To the best of our knowledge, there has been no report on Notch-non canonical Wnt crosstalk regarding the osteo/odontogenic differentiation of hDPSCs. When differentiating into odontoblasts, DPSCs become polarized, which is a prerequisite and fundamental step for cell differentiation49. Non-canonical Wnt is a key signaling pathway that controls cell polarization through the non-canonical Wnt/planar cell polarity pathway50. Thus, we speculate that non-canonical Wnt signaling might participate in the Jagged1-induced osteo/odontogenic differentiation in hDPSCs by regulating cell polarization. However, whether this interaction occurs at the transcriptional level or via elements in the signal transduction chain remains unexplored. In-depth investigations are needed to further reveal the intracellular mechanism between these two signaling pathways.The present study demonstrated the interaction between Notch and non-canonical Wnt signaling. Our results showed that Jagged1 and WNT5A treatment promoted mineralization in hDPSCs. A feedback regulation between these two pathways was observed, as demonstrated by an upregulation of Wnt5A expression by Jagged1 and increased Notch-related gene expression by WNT5A treatment. These findings imply that there might be a reciprocal control between Notch and non-canonical Wnt to regulate hDPSC osteo/odontogenic differentiation. A previous study reported that Wnt5A/serine-threonine Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling mediated Notch signaling activation by interacting with the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), which is a key co-repressor of Notch signaling. CaMKII phosphorylated SMRT, leading to SMRT translocation to the cytoplasm, where it was further degraded by the proteasomeIn summary, the present study found that Notch and non-canonical Wnt signaling had crosstalk during hDPSC osteo/odontogenic differentiation. Notch activation stimulated hDPSC osteo/odontogenic differentiation and promoted WNT2B and WNT5A expression, which suggests the involvement of Wnt signaling in Notch-induced osteo/odontogenic differentiation. Moreover, DKK1 failed to abolish the effect of Notch signaling. However, IWP-2 inhibition of Wnt protein transportation and secretion attenuated the effect of Notch, suggesting that the non-canonical Wnt pathway might partially act downstream of Notch in the osteo/odontogenic differentiation of hDPSCs. Further investigation is required to prove this hypothesis by using specific knockout molecules related to non-canonical Wnt followed by Notch activation to elucidate the specific role of non-canonical Wnt signaling in the crosstalk with Notch in hDPSC osteo/odontogenic differentiation.The hDPSCs were harvested from extracted impacted third molars with informed consent at the Faculty of Dentistry, Chulalongkorn University. The protocol was approved by the Human Research Ethics Committee of Chulalongkorn University  and conducted in accordance with the guidelines of the Biosafety Committee at the Faculty of Dentistry, Chulalongkorn University. Briefly, dental pulp tissues were minced and placed on 35-mm tissue culture dishes for cell outgrowth. The growth medium was composed of Dulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum , 2 mM L-glutamine , 100 unit/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B . The cells were maintained at 37 °C in a humidified 5% carbon dioxide atmosphere. The culture medium was changed every 48 h. Subsequent assays were performed using cells between passages 3 and 7.Calibur flow cytometer using CellQuest software .Surface protein expression was analyzed using flow cytometry. Cells were harvested and prepared as a single cell suspension. The cells were stained with fluorescence conjugated antibodies (1:50 dilution). The antibodies were FITC conjugated anti-human CD44 , PE-conjugated anti-human CD105 , and PerCP-conjugated anti-CD45 . The mean fluorescence intensity was determined by a FACSCells were seeded on 24-well-plates  and maintained in growth medium for 24 h. Subsequently, the medium was changed to osteo/odontogenic medium. The osteo/odontogenic medium consisted of growth medium supplemented with 50 µg/mL ascorbic acid , 250 nM dexamethasone , and 5 mM β-glycerophosphate . In some experiments, the osteo/odontogenic medium was supplemented with either 1, 10, 50, 100, or 200 ng/ml recombinant WNT5A (R&D Systems Inc.) or 500 ng/ml WNT2b (R&D Systems Inc.) to activate Wnt signaling. In contrast, pre-treatment with Wnt inhibitors, 0.5 µg/ml DKK1 (R&D Systems Inc.), and 25 µM IWP-2 (Tocris Bioscience), was used to inhibit Wnt signaling.Cells were seeded on 24-well-plates  and maintained in growth medium for 24 h. Subsequently, the medium was changed to adipogenic medium, which was growth medium containing 0.1 mg/ml insulin , 1 μM dexamethasone , 1 mM IBMX , and 0.2 mM indomethacin . At 16 days, the intracellular lipid droplet accumulation was examined using oil red o staining.11. Briefly, 24-well tissue culture plates were incubated with 50 μg/mL recombinant protein G  for 16 h. Subsequently, the surface was incubated with 10 mg/mL bovine serum albumin solution  for 2 h and then incubated with 0.1, 1, or 10 nM rhJagged1/Fc recombinant protein  for 2 h. Sterile phosphate buffered saline (PBS) was used to wash the culture plates between each step. For the control condition, the human IgG Fc fragment  was placed in the wells in amounts equal to the Jagged1 ligand.Jagged1 was indirectly immobilized on the tissue culture surface as previously describedTo inhibit Notch signaling, the cells were pretreated with a γ-secretase inhibitor, 20 μM DAPT  for 30 min prior to being seeded on the Jagged1-immobilized surface.12. Wnt-related genes were listed according to the KEGG database (entry hsa04310)51. The RNA read counts of significantly different expressed genes were identified using NetworkAnalyst53 and an expression heatmap was generated using Heatmapper54.The high throughput RNA sequencing raw read counts were downloaded from the NCBI Gene Expression Omnibus (series GSE94989)−ΔΔCt method)55. The oligonucleotide sequences of the primers are shown in Supplementary Table Total cellular ribonucleic acid (RNA) was extracted using TRIzol reagent . The quality and quantity of the isolated RNA was examined using a Nanodrop instrument. One microgram of total cellular RNA was converted to cDNA using the ImProm-II Reverse Transcription System . A CFX connect Real-Time PCR  with FastStart Essential DNA Green Master  were used to perform the real-time quantitative polymerase chain reaction. The amplification profile was: 95 °C/20 s, 60 °C/20 s, and 72 °C/20 s for 45 cycles. Melt curve analysis was performed to determine product specificity. The mRNA levels of the target genes were normalized to the 18S gene, and the relative gene expression was quantified with the comparative Ct method  for 30 min at room temperature in the dark. After washing with deionized water, the ALP-stained cells were observed using an inverted light microscope.Cells were fixed with cold methanol for 10 min and gently washed with deionized water. The cells were then stained with a 2% alizarin red s solution  for 3 min at room temperature with gentle agitation. Images of the stained cells were captured using an inverted light microscope. To indirectly measure the amount of calcium deposition, the staining was solubilized with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate at room temperature with gentle agitation for 15 min. The absorbance was measured at 570 nm with a microplate reader .Cells were fixed with 10% buffered formalin for 30 min, followed by incubating with a 0.2% Oil Red O solution for 15 min. Intracellular lipid accumulation was examined using an inverted light microscope .Cells were fixed with 4% buffered formalin at room temperature for 10 min and permeabilized using 0.15% Triton-X100 in PBS. Horse serum (2% v/v) was used to inhibit a non-specific binding. Cells were stained with β-Catenin XP Rabbit mAb  at a 1:100 dilution at 4 °C overnight. Cells were incubated with biotinylated anti-rabbit IgG antibodies  at dilution 1:2000 for 40 min. The targeted protein expression was visualized by stained with Strep-FITC  at dilution 1:500. Nuclei were counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole . Protein expression and localization was detected using a fluorescent microscope with ApoTome system .p < 0.05.All experiments were repeated using cells derived from at least four different donors (n = 4). The statistical analysis was performed using Prism 9 for macOS . The Mann Whitney U test was used for two independent group comparisons. For three or more group comparison, significant differences were determined using the Kruskal Wallis test followed by pairwise comparison. Statistical significance was considered at Supplementary Information.


The genome of retroviruses contains two promoter elements  at the 5′ and 3′ end of their genome. Although the expression of retroviral genes generally depends on the promoter located in the 5′ LTR, the 3′ LTR also has promoter activity responsible for producing antisense transcripts. These natural antisense transcripts (NATs) are a class of RNA molecules transcribed from the opposite strand of a protein-coding gene. NATs have been identified in many prokaryotic and eukaryotic systems, as well as in human retroviruses such as human immunodeficiency virus type 1 (HIV-1) and HTLV-1/2 (human T-cell leukemia virus type 1/2). The antisense transcripts of HIV-1, HTLV-1, and HTLV-2 have been briefly characterized over the past several years. However, a complete appreciation of the role these transcripts play in the virus lifecycle and the cellular factors which regulate their transcription is still lacking. This review provides an overview of antisense transcription in human retroviruses with a specific focus on the MEF-2 family of transcription factors, the function(s) of the antisense protein products, and the application of antisense transcription models in therapeutics against HIV-1 and HTLV-1 in the context of co-infection. NATs are RNA sequences which are transcribed from the opposite strands of DNA with partial or complete overlap with the sense RNA . However, the 3′ LTR (antisense strand) has also been shown to have promoter activity responsible for producing a natural antisense transcript or NAT ense RNA . These NDerived from simian virus transmission, human T-cell lymphotropic virus type 1 (HTLV-1) is the first isolated human retrovirus responsible for pathologies in an infected individual in the form of an aggressive T-cell neoplasm adult T-cell leukemia/lymphoma (ATLL), or a neuroinflammatory disease HAM/TSP  that has similarities to multiple sclerosis . Sense ade novo with the emergence of the HIV-1 strain responsible for the global pandemic  also encodes an antisense protein termed ASP . Antisenpandemic . This annfection . ASP wasnfection . Contrarhbz transcript and the encoded HBZ protein have been shown to promote the survival, proliferation and tumorigenesis of HTLV-1 infected and transformed cells  and HIV-1 coinfection occurs with characteristic presentations . The anted cells .hbz transcript , decreased proviral load, and decreased gene expression. Together, these results suggest that the enhancer element alone does not determine persistence and disease development but plays a critical role in regulating viral gene expression in the infected cell.DNA elements within the HTLV-1 provirus, including a viral enhancer  and the nfection . Quite rnfection . Cellulanfection . The losin vitro . Howeverin vitro and reduction of proviral load in a humanized mouse model. These studies provided a novel mechanism of 3′ LTR regulation and established MEF-2 signaling as a potential target for therapeutic intervention for ATLL. MEF-2C was found to be highly enriched at the 3′ LTR along with cofactors Menin and JunD resulting in binding of MEF-2C to HBZ at this region. JunD is critical for the regulation of both HIV-1 and HTLV-1 3′ LTR -2 that was previously established to have a role in HTLV-1 pathogenesis . More re1 3′ LTR , and upr1 3′ LTR . MEF-2C 1 3′ LTR , several1 3′ LTR  that neeTypical expression of MEF-2 isoforms occurs in almost every human tissue, ranging from hematopoietic stem cells to the cerebral cortex. These MEF-2 isoforms play key roles in controlling cellular differentiation and maintenance of mature cell types. MEF-2 isoforms, mainly MEF-2A and -2C, have also been implicated in various leukemias and lymphomas, as well as virus infection (reviewed in de novo DNA methylation by preventing H3K9 methylation within the same region (H3K9 methylation is a repressive modification that may serve as a molecular beacon for certain DNA methyltransferases). Interestingly, MEF-2C has been shown to interact with the chromatin architecture of schizophrenia and improves cognition in mice  for antiretroviral therapy . Prior t benefit . However benefit . Even grin vitro . Two gRNTherapeutic applications of CRISPR technology extend to HIV-1 as well. For some time, the concept of using CRISPR gene editing as a therapeutic intervention for HIV-1 infection has been considered as a key research point. While highly active anti-retroviral therapy (HAART) drugs have remained the mainstay of HIV-1 treatment, these drugs act by suppressing HIV-1 replication, and do not effectively eradicate HIV-1 proviral DNA from latently infected cells. Once HAART therapy is ceased, the latent infection is able to rebound and continue to debilitate patients . Thus, tin vitro and decreased proviral load in vivo. MEF-2C also plays a role in several other leukemias and lymphomas. Notably, it plays a role in EBV oncogenesis and in the proliferation of EBV-induced lymphoblastoid cell lines. Disruption of MEF-2C and other transcription factors binding to “super enhancer” regions disrupts EBV oncogenesis.Retroviruses such as HIV-1 and HTLV-1 contain LTRs at both ends of their genome. While it is well established that retroviral gene expression is dependent mainly on the 5′ LTR, antisense transcription originating from the 3′ LTR produces mRNA and protein products that have a great deal of influence on the course of retroviral infection in both HIV-1 and HTLV-1 (summarized in A possible avenue of investigation and therapy exists with CRISPR technology. There is potential in both identifying transcription factors that bind to the 3′ LTR in HTLV-1, as well as in a wider set of therapeutic applications involving knock-out of key genes for viral proliferation, or knock-in of therapeutic genes or gene mutations. The understanding of antisense transcription in retroviruses is still in its infancy. Much remains to be discovered regarding the roles of antisense transcripts and their protein products in the regulation of retroviral life cycles, as well as virally induced oncogenesis. There is a potential for new therapeutic strategies against both retroviral infection as well as their oncogenic endpoints.EL and AP equally contributed to writing of the manuscript. GS contributed to writing of the manuscript. PJ conceived the idea, assisted with writing of the manuscript, edited text, and figure drafts. All authors contributed to the article and approved the submitted version.via R01 NS097147 to PJ.Authors acknowledge funding support from the NIH/NINDS 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.


The Joint United Nations Program on HIV/AIDS (UNAIDS) has adopted the Sustainable Development Goals (SDGs) to end the HIV/AIDS epidemic by 2030. Several factors related to the non-suppression of HIV, including interruptions of antiretroviral therapy (ART) and opportunistic infections could affect and delay this projected epidemic goal. Human T-Cell leukemia virus type 1 (HTLV-1) appears to be consistently associated with a high risk of opportunistic infections, an early onset of HTLV-1 and its associated pathologies, as well as a fast progression to the AIDS phase in co-infected individuals, when compared to HIV-1 or HTLV-1 mono-infected individuals. In Gabon, the prevalence of these two retroviruses is very high and little is known about HTLV-1 and the associated pathologies, leaving most of them underdiagnosed. Hence, HTLV-1/HIV-1 co-infections could simultaneously imply a non-diagnosis of HIV-1 positive individuals having developed pathologies associated with HTLV-1, but also a high mortality rate among the co-infected individuals. All of these constitute potential obstacles to pursue targeted objectives. A systematic review was conducted to assess the negative impacts of HTLV-1/HIV-1 co-infections and related factors on the elimination of HIV/AIDS by 2030 in Gabon. The human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) pandemic reduces the availability of human capital and negatively affects the economic growth worldwide . HIV/AIDAn estimated 38.4 million people are affected by HIV-1, with approximately 1.5 million new infections diagnosed annually, accounting for 650,000 deaths, according to the UNAIDS report. The epidemiological situation of HIV-1 worldwide, by region, showed that the region of sub-Saharan Africa was the most affected with approximately 25.6 million people living with HIV (PLHIV) for 860,000 new cases and 420,000 AIDS-related deaths annually [The HIV-1 infection in Gabon is characterized by an important genetic diversity that includes the circulating groups, sub-types, and all recombinant forms of HIV. As group M is the most circulating in the country, the sub-type A, and the recombinant form CRF02_AG were found to be predominant. These HIV-1 variants were described by a general population, specifically in Libreville and Franceville, the capital city of Gabon and the province of Estuary, and the capital of the province of Haut-Ogooué (southeast), respectively . These fThe HIV screening in Gabon is based on the performance of several serological laboratory tests. The tests are performed according to the recommendations of the national program for the fight against HIV/AIDS and sexually transmitted infections , thus combining the detection of antibodies directed against HIV-1 and HIV-2, and the detection of antigen (Ag) p24. The recommended tests are the DETERMINE HIV1/2, Alere COMBO HIV, and SD Bioline HIV-1/2 3.0 . These rAccording to the manufacturers, all three tests have 100% sensitivity and 99.8% specificity levels. As per the manufacturer’s recommendations, these tests can detect all HIV-1 subtypes, which are predominant in Gabon. A recent study carried out in Libreville, evaluated the Alere COMBO HIV and DETERMINE HIV1/2 tests, and the results confirmed the stipulated sensitivity while the specificity for Alere COMBO HIV and DETERMINED HIV1/2 were 94.6% and 98.6%, respectively . Based oThe Joint United Nations Program on HIV/AIDS (UNAIDS) has set goals of 95-95-95 targets for eliminating HIV/AIDS by 2030. The final 95 means that 95% of people on an ART with a suppressed viral load, is only possible if 95% of PLHIV are on ART. Indeed, treatment is the main mean by which these goals can be achieved . HoweverEarly epidemiological studies on the HTLV-1 infection in Gabon were conducted by Delaporte et al. in 1986. The initial study group consisted of 2558 participants made of 1874 adults and 684 children from cities of five provinces including the northeast province of Ogooué-Ivindo, the southern province of Ngounié, the southeastern province of Haut-Ogooué, and the major cities of Libreville and Port-Gentil of the coastal and western provinces of Estuary and Ogooué-Maritime, respectively . An indiAdditional studies were performed and confirmed the previous findings. In 1993, the first epidemiological data on the HTLV-1 infection were obtained after analyzing blood donors and pregnant women collected at the Regional Hospital of Franceville, Gabon. Six-hundred-and-thirty-three pregnant women and seven-hundred- and four donors were tested with ELISA and the positive samples were confirmed by a western blot (Dupont de Nemours). The HTLV-1 positivity was defined as the presence of gag (p19 with/without p24) and the envelope glycoprotein gp46. The western blot tests were performed with less serologically stringent criteria positivity, to better compare the characteristics with those of other studies previously conducted in the same region. Using less serologically stringent criteria lowered the risk of having negative results for the possible HTLV-2 positives and/or other specific STLV variants from Gabon. The reactivity to p19 and/or p24 with at least one pX gene protein encoding for the tax protein  or gp46 was detected and the HTLV infection was confirmed with a seroprevalence rate of 9.3% among pregnant women and 10% in blood donors . That suenv and LTR region), an overall prevalence of the HTLV-1 infection rate of 8.7% among the rural population was reported and linked to the 8.5% previously reported by Hlela and coworkers in 2009, after compiling the epidemiological HTLV-1 infection data in Gabon, from 1980 to 2007 [gag, pol, and pX (coding for the tax and rex proteins) regions, the HTLV-1 prevalence rate was estimated at 3.7%, compared to 8.1% for the seroprevalence rate [An investigation by Hersan and coworkers aimed to identify the demographic and social factors involved in the high HTLV-1 seroprevalence observed in Haut-Ogooué province, demonstrated in 1994 with a predominance of the HTLV-1 infection in female individuals together with heterogeneous seroprevalence, according to the ethnics groups, and with the highest prevalence obtained in the Kota-Obamba group, unlike the Teke group (11% vs. 3.4%) . The dif to 2007 . The sam to 2007 ,41,43. Hnce rate . This conce rate . This reOne of the latest epidemiological studies on the HTLV-1 infection in Gabon, was conducted on 4381 individuals from 220 villages spread over all nine provinces of the country. The heterogeneity of the HTLV-1 prevalence, according to the provincial areas was defined as follows: a relatively low prevalence (2–5%), a strong prevalence (5–10%), and a very strong prevalence or endemic prevalence (rate ≥ 10%). From the serological tests (ELISA and western blot), together with the molecular tests through the PCR, an overall HTLV-1 prevalence rate was estimated at 7.3%, which was very close to 8.7%, reported by Djuicy and coworkers in the same year ,12. ThatBeyond the studies based on the HTLV-1 epidemiology, many other possible factors associated with the HTLV-1 infection have been investigated, and the vertical transmission of this retrovirus is of particular interest. A particular study demonstrated that there is no association between the HTLV-1 infection and the infertility status, while the seroprevalence rate of the HIV-1 infection was mostly higher in women with primary infertility, than others (9.3% vs. 0.7%) . These pn = 302) against 0.7% (n = 574) for the HTLV-1 prevalence in the Taï region, in the Cote d’Ivoire. This trend seems to be inversed for the STLV-1 infection for these two regions with 23% (n = 39) and 39% (n = 111), respectively, in each region. Remember that here, the aim of Mousson’s study et al. was to evaluate whether frequent contacts between human and NHP bushmeat infected by STLV-1, in the region, could be related to the HTLV-1 prevalence in the same region. Following a multivariate analysis, the authors highlighted the higher frequency butchering activities of NHPs in the Democratic Republic of the Congo, relative to the Cote d’Ivoire region. Furthermore, they reported two HTLV-1 strands very closely related to STLV-1, isolated from same region in the Cote d’Ivoire [In addition to maternal transmission, blood transfusion could represent one of the major routes for spreading HTLV-1 in Gabon. Indeed, as it was said above, in 1993, Berteau et al. reported a 6% HTLV-1 seroprevalence rate among 704 blood donors in Franceville in Haut-Ogooué province. In 2017, a similar study was carried out in Libreville at the National Center of Blood Transfusion (CNTS), by Ramasamy et al., where they found a 0.74% HTLV-1 prevalence rate from 3123 blood donors . Those sd’Ivoire . Thus, tGabon is one of the most endemic countries for the HTLV-1 infection in the world, with about 16,000 to 30,000 infected people according, to Gessain and Cassar’s published work, in 2012 ,61. AbouEnv gene, the new Gabonese strains were classified into a new group (subtype D) of African strains [tax, env, and LTR genes of the HTLV-1 strains from pregnant women or blood donors found in the HTLV-1 strains of subtype B, but also one belonging to the cosmopolitan subtype A [Papio genus).The first molecular biology analyses on the strains of HTLV-1 began in 1987. Following the culture of lymphocytes from a healthy HTLV-1 carrier, the provirus was analyzed using a southern blot. The provirus genome of the studied strain showed about 38% of differences between its restriction sites and those of the Japanese prototype. In addition, its restriction map looked similar to that of a STLV-1 (isolated from a Sierra Leonean chimpanzee) and a B subtype HTLV-1 from Zaire . These r strains . The sequbtype A ,68. Thusubtype A ,48. Thisubtype A . In addiubtype A . An inteubtype A . This HTFinally, another study on the whole sequences of the envelope genes, compared with all of those of PTLV-1 available in the GenBank, showed that the Gabonese strains HTLV-1 belong to subtypes B of Central Africa, cosmopolitan A and with characteristic groups, according to the mutations observed . The phy3) than in the HIV-1 mono-infected (481.0 ± 299.0 cells/mm3) individuals. Similarly, the mean HIV-1 viral load was Log 3.0 ± 1.6 copies/mL in mono-infected individuals and Log 2.3 ± 0.7 copies/mL in co-infected individuals.Despite the description of both retroviruses, only one study was recently published on the co-infections in populations living in Gabon . A totalThe targets of the WHO, relative to epidemiological surveillance and HIV eradication by 2030 through the 95-95-95 program, could be hampered by some infections, such as HTLV-1, which have the ability to counteract and negatively modulate the survival prognosis of PLHIV ,74. HTLVMycobacterium tuberculosis infection  in HIV-1/HTLV-1 co-infected individuals, compared to the prevalence of the same infection in HIV-1 mono-infected individuals [Although several studies are reporting both a faster progression to the AIDS phase and the often-higher CD4+ count in HIV-1/HTLV-1 co-infected people, compared to HIV-1 mono-infected individuals alone, nothing seems to be solved about these observations ,79,80. Hividuals ,87. Moreividuals ,89,90. Vividuals . Despiteividuals . Severalividuals . Howeverividuals ,94. As tividuals , the intividuals , thus pr6 peripheral blood mononuclear cells (PBMCs) and a HIV-1 viral burden load < 10,000 copies/mL [A study by Beilke et al. investigating the relationship between the HTLV-1/2 viral load, clinical parameters in HIV-1 positive patients on ART and its co-infection with HTLV-1 or HTLV-2, found a significant association between the HTLV-1 burden proviral DNA > 20,000/10opies/mL . This stSeveral studies have shown a high prevalence of HTLV-1 among HIV-1 infected people, compared to the general population in endemic areas, for both retroviruses. HTLV-1-induced pathologies remain unknown to most medical practitioners. Thus, people living with a disease associated with HTLV-1, could potentially be positive for HIV-1 but could go unnoticed, constituting the first obstacle in the aim of the WHO for 2030. However, many others studies demonstrated the HTLV-1 ability to activate the HIV-1 viral latency, while others reported a high level of proinflammatory cytokines production and a high HTLV-1 viral load in HIV-1/HTLV-1 co-infections, compared to HIV-1/HTLV-2 or HIV-1 and HTLV-1/2 mono-infections. These results highlight the involvement of these co-infections in the worsening of symptoms and the immunosuppression, even on ART, the faster development of the HTLV-1 diseases, and the progression to the AIDS state. Furthermore, in Gabon, as for other endemic areas for both retroviruses, a systematic diagnosis of HTLV-1 should be imposed on HIV-1 positive patients (and vice versa) to evaluate their survival prognosis. In addition, to achieve the 2030 WHO goals, in the endemic area, a systematic screening of HTLV-1 should be imposed, as well, on pregnant women, and blood transfusion should be avoided or limited, as it increases the risk of HIV-1/HTLV-1co-infections. Finally, the use of condoms during sexual intercourse, and screening organs before transplantations must be also implemented to strengthen the fighting strategy against both viruses. These would help in achieving the WHO goals, as the main ways proposed for the HIV-1 elimination by 2030 could be used for the HTLV-1 elimination as well. considering the high contact frequency between humans and NHPs (causes of bites), and the NHP consumption in Gabon, a sensibilization campaign about the risks involved could appear as an important effort to achieve the aims of the WHO, by 2030.