Single-nucleotide polymorphisms (SNPs) in the p53 pathways have shown to play a role in endometrial receptivity and implantation in infertile women undergoing in vitro fertilization (IVF). The present study aimed to assess the influence of these gene variants over pregnancy success through a receptivity model in recipients of egg donation treatments, when factors such as age and quality of the oocytes are standardized.TP53 Arg72Pro (rs1042522), LIF (rs929271), MDM4 (rs1563828), and USP7 (rs1529916) SNPs in the recipients allowed comparison of allele and genotype frequencies and their association with the IVF treatment outcome.A nested case–control study was performed on 234 female patients undergoing their first fresh IVF treatment as recipients of donor oocytes. Genotyping of USP7 (rs1529916) and MDM4 (rs1563828) were associated to differential ongoing pregnancy rates and variable miscarriage events, respectively.Grouped by genotypes, patients showed differences in IVF outcomes after the embryo transfer. Arg72Pro (rs1042522) gene variant was associated to changes in implantation and clinical pregnancy rates. The polymorphisms MDM4 (rs1563828) and USP7 (rs1529916) gene variants were associated with the later maintenance of pregnancy.This study highlights the association between gene polymorphisms related to P53 function and their influence over IVF reproductive outcomes. Arg72Pro variant may influence early events, as lower implantation rates were found in homozygous for Pro72 allele. By contrast,  Pregnancy establishment requires important cellular and molecular changes through differentiation and proliferation processes . SeveralLIF) [TP53 has been highlighted in embryo implantation, as the processes involved require intense transformation of cells that are additionally exposed to a very changing environment. It is thus suggested that a protective role of p53 could be relevant for maintaining the cells’ genomic stability at the embryo-maternal interface [The P53 family comprises a wide number of hyperconnected transcription factors associated to cellular apoptosis, cell cycle arrest, senescence, and DNA repair. The control of these processes by P53 protein products characterizes their function as a tumor suppressor –7. Due tLIF) . It has LIF) , 10. Thenterface .LIF [LIF participates in many diverse biological processes in the uterus involving cell proliferation, differentiation, decidualization, immune response, and vasculature remodeling [LIF levels in the endometrium could impair the establishment and maintaining of the pregnancy and that elevated and maintained LIF levels may improve endometrial receptivity [The influence of the P53 family in reproduction has been proposed to be dependent on LIF , whose cLIF –15. LIF modeling . Currenteptivity , 17.Additional gene variants related to the P53 pathway have been associated to reproductive success –20, and For this study, we used the model of pregnancy establishment in women undergoing treatments using donor oocytes, to achieve a receptivity model for the investigation of biological features regarding embryo implantation when the recipient does not share genetics with the embryo to be implanted and to minimize the effect of factors intrinsically attained to oocyte competence, such as age.TP53; the rs929271 of LIF gene, whose function is essential during embryo implantation and which also seems to regulate P53 [MDM4) whose protein product inhibits P53 activity [USP7) gene that shows a splicing variability according to genotype [For this genetic association study, we settled a list of candidate single-nucleotide polymorphisms (SNPs) related to P53 pathway, and these were genotyped to explore the reproductive outcome of IVF treatments in women recipient of donor oocytes. The selected variants consisted of the most known functional variant rs1042522 (Arg72Pro) in late P53 , 25; theactivity ; and thegenotype , as its genotype .2, or if there was severe male factor infertility in the partner. The patients provided a signed consent form for the IVF treatment and also for the genetic study and provided a DNA sample obtained from buccal swab.A genetic association study with a nested case–control design was performed to assess the effect of selected candidate gene variants related to P53 pathway on the outcome of oocyte donation treatments and pregnancy progress. Patients undergoing IVF with donated oocytes were recruited for the present study at a private IVF unit. Included women were European Caucasian aged between 18 and 45 years undergoing the first oocyte donation treatment as recipients and receiving a fresh embryo transfer (ET) of at least two good-quality cleavage-stage embryos on day 2 or day 3. Women were excluded if they presented with uterine factor infertility, abnormal endometrial thickness, suspected hydrosalpinx, body mass index (BMI) > 30 kg/m2. Donors were screened for known hereditary disorders, chromosome abnormalities, and infectious diseases. Donors were not accepted if they previously presented an abnormal response to gonadotropin, polycystic ovary syndrome (PCOS), endometriosis, more than one previous miscarriage, and any other medical condition that could compromise the success of the treatment.Oocyte donors were European Caucasian healthy women with good physical and mental health aged 18–35 years, having regular menstrual cycles and a body mass index between 18 and 29 kg/m2) and 6% (CO2) using sequential commercial culture medium cleavage and blastocyst (SAGE).Human recombinant follicle-stimulating hormone (hrFSH) and gonadotropin-releasing hormone (GnRH) antagonists were used for COH in donors, and hrFSH units were adjusted between 150 and 300 IU depending on age, BMI, antral follicle count (AFC), and ovary response. Oocyte maturation was triggered by administering GnRH analogue after having reached at least three dominant follicles ≥ 17 mm. Oocyte retrieval was done 35 h after the GnRH analogue administration. Denudation of cumulus oocytes complexes was performed by mechanical disaggregation after a brief exposure of hyaluronidase (40 IU/mL) in HEPES. Mature metaphase II oocytes obtained were inseminated using sperm from the male partner through intracytoplasmic sperm injection (ICSI) 2 h after denudation. Embryo culture was performed in low oxygen atmosphere 5% (OEmbryo transfer (ET) to the recipients was performed on day + 2 or day + 3. Recipients underwent endometrial preparation based on suppression of ovarian function and increasing dosage of estradiol valerate tablets. From the day of donor’s oocyte retrieval, the recipients received micronized progesterone (P) vaginal pessaries for luteal phase support. The ET was performed only after confirmation of an endometrium with typical trilaminar pattern with a thickness of at least 6 mm. Embryos were selected for ET when at least two good-quality embryos reached grade A or B following Spanish Society for Studies on Reproductive Biology (ASEBIR) .Pregnancy was determined by serum or urine tests detecting positive levels of human choriogonadotropin hormone β-hCG in oocyte recipients after two consecutive measurements in day + 11 or + 12 after ET, depending on cleavage stage of transferred embryos day + 3 or + 2, respectively (β-hCG ( +)). Clinical pregnancy (CP) was determined by ultrasonographical evidence of a gestational sac 5–6 weeks after ET. The absence of an ultrasound-identifiable pregnancy and a decrease in the levels of β-hCG represented a biochemical pregnancy loss (BPL). An ongoing pregnancy (OP) was defined as the presence of at least one viable fetus after week 12 of gestation. Miscarriages (M) were considered to be those gestational losses that occurred between weeks 4 and 20, having previously identified a gestational sac . The impTP53 (rs1042522), LIF (rs929271), MDM4 (rs1563828), and USP7 (rs1529916). Allelic assignment was done using TaqMan Genotyper® . Positive and negative controls were included in each reaction assay to ensure the quality and concordance of results to validate them. A minimal calling rate of 80% was used to assign genotype in the SNP. The list of gene variants included in the panel is summarized in Table Genomic DNA was isolated from buccal swab collected from the recipients and using the QIAamp DNA Mini Kit . Genotyping process was outsourced to the genetic and proteomic services of Science and Technology School in the University of Pais Vasco (Bizkaia). TaqMan® Open Array Genotyping System  was used to identify TP53 rs042522, LThe study was designed as a nested case–control in which cohort groups are established according to the results obtained after the IVF treatment. This enables the possibility of analyzing the incidence of the genotypes according to the population stratified by treatment results and by pregnancy progress. Positive or favorable outcome was settled as control and negative outcome as a case.To assess any possible population bias compared to the general population, allele and genotype frequencies published for the studied SNPs in 1000 Genomes (Phase 3) were used as reference . Differep < 0.05 2-sided for all the tests.The clinical treatment outcomes IR, CP, BPL, M, and OP were used to assess the treatment progression and also their variation between recipients grouped by genotypes through Fisher’s exact and chi-squared tests. When a variation in the number of recipients was present on any group representing a clinical significance, a logistic regression model was used to compute the odds ratio with a 95% confidence interval, and the age of the recipient was used as covariate and treated as a potential confounder for adjustment. Groups of genotypes for comparison were established by considering the possible recessive, dominant, codominant, and over-dominant models of inheritance. To provide the fittest model of interaction between genotypes, and assuming a total absence of a known direct biological influence, Akaike’s (AIC) and Bayesian’s (BIC) information criteria were used to select the degree of association for every genotype with treatment outcomes . StatistA total of 234 Caucasian European recipients undergoing an IVF treatment with donated oocytes aged 40.48 (± 4.37) were included in the study (Table MDM4 gene variant (rs1563828) (Table MDM4 variant in the non-pregnant recipients (p = 0.006) , and the only differences were observed for the p = 0.016) compared to β-hCG ( +), which resulted in an increase of homozygous Pro72 (C/C) recipients in the non-pregnant recipients, compared with the β-hCG ( +) group (Table USP7 (rs1529916) gene variant was observed in β-hCG ( +) patients  (C) showed a significant 12% increase in recipients tested β-hCG ( −) (up Table . A signits Table . All thets Table .p = 0.047); in the same line, these recipients also presented significantly reduced CP (38.89%) compared with the Arg72/Pro72 heterozygous (58.67%) and the Arg72 homozygous (68.42%). Recipients grouped by genotypes of the MDM4 (rs1563828) and USP7 (rs1529916) presented differences in the miscarriage and also in OP rates, respectively  was associated to increased OP under a dominant model (Table MDM4 (rs1563828), and heterozygous A/G women presented with fewer miscarriages  was associated to higher likelihood of achieving successful embryo implantation and pregnancy establishment. Our study suggests that certain clinical outcomes could be associated to USP7 (rs1529916) and MDM4 (rs1563828) gene polymorphisms, which might affect the following events for maintenance of an ongoing pregnancy.The present study used a model based on donated oocytes to investigate the association between p53-related gene variants and the success of IVF treatments in women recipients of donor eggs. Our results showed that USP7 gene variant (rs1529916) was associated with later pregnancy events, and recipients carrying at least one A allele had higher OP rates than homozygous G/G. However, it is very interesting that the homozygous genotype A/A, which represents the lowest frequency not only among the studied population but also in the wide European people (1000 Genomes) [USP7 variant might also be associated with earlier events during pregnancy establishment and not only with events in the later stages. A lower risk for miscarriage was observed for heterozygous of the MDM4 gene variant. These results reinforce the idea about the potential utility of p53-related gene variants as predictive markers in IVF and more specifically as an indicator of receptivity potential.The group of recipients that did not achieve pregnancy showed a significant increase in the frequency of the allele Pro72 Table , and IR Genomes) , signifiIn this study, we did not screen for abnormal chromosomal errors in the embryos, which is a limitation of the study; however, to minimize this potential impact, our study was designed using the model of egg donor treatment, where donors are young and healthy women. Our results are in agreement with previous genetic association studies reporting a negative impact of Pro72 homozygous genotype on IVF treatments outcomes , 22, 24.The available literature on possible associations between p53 gene variants and IVF outcome have so far had little or null impact in the practice of reproductive medicine. A main reason may be dependent on the previous inconsistent results reported  or that Our study aimed at investigating the impact of these gene variants of p53 on IVF outcomes, however using a specific model with good expected oocyte competence from donors and reduced dependency of female age factors, where the model of implantation and pregnancy establishment would be less impacted by these bias. Studies on IVF treatments in oocyte recipients have been previously conducted, but little information has been provided regarding the inclusion criteria of recipients and the selection criteria and age limit of the oocyte donors . The recMDM4 and USP7 are highly expressed in the endometrium, and they are critical regulators affecting P53 activity [MDM4 variant and lack of pregnancies has to be interpreted with caution, as it could be due to induced bias, as it did not accomplish HW equilibrium in the recipients that did not achieve pregnancy.The genes activity , 34, 35.activity . The datLIF expression indicates that it is two times higher in Arg72 homozygous carriers compared to that in Pro72 homozygous, which could impact differently success of pregnancy establishment [LIF functional expression that has been estimated for these genotypes [LIF levels, and this could impair cellular and molecular dialogue during early embryo implantation, however without clear influence on the later progress of the pregnancy. By contrast, USP7 (rs1529916) and MDM4 (rs1563828) gene variants may have an impact in later stages. At present, the functional implications of these intronic changes are unknown, but it could be hypothesized that perhaps they could be linked to any other functional variant or at least mediate, under a genomic regulatory mechanism that potentially modify p53 activity and/or levels, in a manner that may impair the pregnancy maintenance.Biological processes by which p53 could mediate embryo implantation remain unknown. LIF has been postulated as a mediator of the p53 pathway, and it has been shown to regulate embryo implantation in mammals –38; howelishment . Our resenotypes . Our resLIF or modification of p53 levels and/or activity could be a potential alternative to explore new research or future clinical improvements [Our data add to the current literature and our observations merit further investigation of the influence of p53 on embryo implantation and evaluate its predictive value for the clinical management of patients undergoing IVF treatments. As proposed by previous authors, controlled therapies based on exogenous administration of ovements .The present study provides support of a clinical association between polymorphisms located at genes involved in the P53 pathway and the success of IVF in recipients of donor oocytes, which was used as a model of pregnancy establishment independent of the oocyte genetic origin and reducing the bias of poor oocyte competence. Further studies are needed to clarify the biological context of our findings and to extend the knowledge of polymorphism of genes of the P53 pathway in different populations and ethnicities.


BackgroundUnderrepresented-minorities (URM) remain few in number amongst practicing cardiologists and across cardiology fellowship training programs in the U.S. Increased diversity is needed across the entire field and is particularly necessary within graduate medical education cardiology fellowship training programs.ObjectivesThis cross-sectional study was performed to identify which strategies were supported and implemented by cardiology fellowship program directors (PDs) to increase URM representation, to determine which entities hold the responsibility to increase diversity according to program directors, and to quantify URM representation in cardiology fellowship programs.MethodsA 15-item survey was submitted to all American College of Graduate Medical Education (ACGME) accredited cardiology fellowship programs via electronic mail.ResultsOf 250 cardiology fellowship programs, 71 responses were received (28.4%). The number of URM faculty varied from 0-1 to more than six, and URM faculty held leadership roles in most programs (62.0%). A total of 16 respondents (22.5%) were URM program directors. Most respondents agreed that diversity was important to their training program (85.9%). The majority endorsed direct recruitment of URM applicants (60.6%), opportunities for applicants to connect with (54.9%) or be recruited by URM fellows (54.9%), holistic application review (67.6%), promoting mentorship by URM faculty (69.0%), URM faculty involvement in applicant interviewing (54.9%), and increased recruitment of URM faculty members (73.2%). Program directors allocated major responsibility to increase diversity to fellowship programs (71.8%), residency programs (63.3%), and medical schools (53.5%).ConclusionsThis study found that most cardiology programs have URM faculty in leadership roles, and nearly a quarter of the surveyed program directors were URMs. Cardiology program directors endorsed and employed numerous strategies to increase diversity and URM representation in fellowship programs. Additionally, program directors held fellowship training programs most responsible for increasing URM representation in the field of cardiology. Underrepresented-minority (URM) physicians, specifically African American, Native American, Hispanic, and/or Pacific Islander, constitute only about 10% of practicing cardiologists . DiversiA review of data from the past decade has shown progress in increasing the number of cardiology trainees who would be considered URMs. However, URM cardiologists remain a considerable minority in the cardiologist workforce nationally. In fact, the percentage of URM cardiologists increased only modestly from 11.1% to 12.4% from 2006 to 2016, respectively . In addiGiven that fellowships constitute the entryway into the field of cardiology, the role of the fellowship program director (PD) in increasing diversity in the cardiologist workforce cannot be overstated. A 2016 survey by Crowley et al. reported that 63% of fellowship program directors believed that diversity in their program was adequate and did not require further intervention; this study also reported that 69% of the same respondents agreed with the statement, “diversity is a driver of excellence in healthcare delivery” . AnotherThis survey of cardiology fellowship program directors builds upon previous research in several unique ways. This study was structured to quantify URM applicants, fellows, and faculty members in ACGME-accredited cardiology fellowship programs and identify which strategies are supported and currently implemented by program directors. Moreover, this study seeks to determine which entities are viewed by fellowship directors to have a role in increasing diversity in cardiology training programs.Fellowship program database constructionThe University of Southern California Biomedical Institutional Review Board (BioIRB) determined this study to be exempt (#HS-20-00319). After receiving this IRB approval, we conducted an anonymous cross-sectional study of 250 ACGME-accredited cardiology fellowship program directors in the United States. The following data were collected: program name and location, director full name, program director email address, and program coordinator email address. Data were abstracted from the Fellowship & Residency Electronic Interactive Database (FREIDA) and cross-referenced with the ACGME website. If the program director's contact information was unavailable, we attempted to obtain this information through internet searches of faculty directories, corresponding author contact information , and scientific databases .Survey development and disseminationSurvey questions were created through an iterative process by members of the research team & diversity content experts and by referencing previous surveys created to assess diversity in medical fellowship programs . The surStatistical analysisDescriptive statistics were applied to the dataset. Fisher’s exact tests were used to compare the number of respondents that supported each strategy to increase diversity to respondents that actively implemented those measures. Predictors of strategies implemented by PDs to increase diversity were identified a priori: URM status and agreement with the statement “diversity is important to my residency program.” We then dichotomized agreement regarding the importance of diversity into two variables prior to comparison: PDs that “strongly agreed” versus PDs that “somewhat agreed,” “neither agree nor disagreed,” “somewhat disagreed,” and “strongly disagreed” with the above statement. The latter group was hereby referred to as “not strongly agreed” in ensuing data comparisons. Fisher’s exact tests were used to (1) compare strategies that were implemented by respondents who identified themselves as a URM to respondents who identified themselves as non-URM members, and (2) compare strategies that were implemented by PDs that “strongly agreed” versus those who did “not strongly agree.” P-values less than 0.05 were considered statistically significant.A total of 71 responses were obtained from 250 ACGME-accredited cardiology fellowship training programs, constituting a 28.4% response rate. The total number of fellowship training positions available ranged from 3 to 35, with an average of 15.3 positions per program (standard deviation (SD) = 6.8). Cardiology fellowship training program characteristics are described in Table URM representation in cardiology fellowship programsThe number of URM fellows ranged from 0 to 20, with an average of 4.5 URM fellows per program (SD = 3.8). Of interviewed applicants, the number of URM matriculants ranged from 0 to 4, with an average of 1.3 (SD = 1.1). The reported number of URM faculty per program included 0-1 , 2-3 , 4-5 , and 6 or more . URM faculty members reportedly held leadership roles in the majority of fellowship programs , although respondents from two programs were unsure if URM faculty held leadership roles (2.8%). Of 71 program directors, 22.5% (n = 16) considered themselves URM. A detailed analysis of URM representation in cardiology fellowship programs is described in Table Outreach strategies to URM residentsRespondents’ support of strategies to increase fellowship diversity and current, active implementation of outreach to URM residents is described in Table Modifications of URM application reviewStrategies to address diversity through changes to the application process that are supported by and implemented by cardiology fellowship directors are indicated in Table The use of diversity-related programmingThe numbers and percentages of fellowship directors that support and implement various diversity-related programming are described in Table Support systems for URM fellows & diversity initiativesStrategies of active promotion of diversity within cardiology fellowship training programs according to program directors are described in Table Comparing responses of URM program directors vs. non-URM program directorsOf 71 program director respondents, 22.5% (n = 16) identified themselves as URM members, while 77.5% (n=55) identified themselves as non-URM members. A statistically significant difference was found regarding the involvement of current program fellows in informal recruitment of URM applicants (p =0.0007) as 70.9% (n=39) of non-URM program directors implemented this strategy compared to only 31.3% (n=5) of URM program directors. No other statistically significant differences were found in the implementation of any other strategies between URM PDs compared to non-URM PDs (p > 0.05).Implementation strategies of program directors that “strongly agree” vs. “not strongly agree” that diversity is important to their fellowship programComparative responses of diversity strategies that are currently being implemented by respondents who responded as “strongly agreed” (n=49) to the statement, “diversity is important to my residency program,” were contrasted against program directors who responded with any other response (n=22) (“not strongly agree”). Despite similarities between the groups, a statistically significant difference was found between the groups when implementing a holistic review of URM fellowship applicants (p = 0.028) as program directors that “strongly agree” with the importance of diversity implemented this strategy at a rate of 77.6% (n=38), while only 50.0% (n=11) of program directors that placed lesser importance on diversity implemented this strategy. No other statistically significant differences were found in the implementation of any other strategies between “strongly agree” PDs compared to “not strongly agree” PDs (p > 0.05).Whose responsibility is it to increase diversity in cardiology fellowships?Responsibility for increasing diversity according to fellowship program directors is described in Figure This cross-sectional study was performed to assess the strategies endorsed and implemented by cardiology fellowship training program directors and the representation of URM applicants and faculty in ACGME-accredited programs. This study found that the vast majority of PDs expressed agreement with the view that diversity is important in fellowship training programs (85.9%). However, PDs remain unsure how to implement the desire for diversity in the process of selecting candidates and creating a rank list . In factFurthermore, this study showed that PDs tended to support several different methods to increase URM representation and implemented these strategies to increase diversity in their program. These findings suggest that program directors believe that they have the means to employ methods that meaningfully increase diversity within their programs. As in previous studies, most fellowship directors claimed to support diversity, although a previous study found that less than half of directors reported having a formalized plan to achieve this goal -12. AchiIn order to achieve a higher degree of diversity and inclusion, cardiology fellowship directors largely expressed support for increasing the number of URM faculty members (73.2%). It has previously been shown that the presence of URM faculty, mentorship of URM faculty with URM fellows, and communication between URM faculty and fellows with URM applicants on interview day is critical to recruiting competitive, diverse cardiology fellows . In our Implementation of diversity strategies by URM program directors was similar compared to that of non-URM directors, with the exception of involving current program fellows in the informal recruitment of URMs. Non-URM members overwhelmingly implemented this strategy at a rate of 70.9% (n=39) compared to only 31.3% (n=5) URM program directors. As previously described by Auseon et al., having URM leadership in cardiology fellowship programs is often a “rate-limiting step” to attracting URM fellows to a program . As suchProgram directors that “strongly agreed” that diversity is important to their program implemented diversity strategies at similar rates compared to program directors who did “not strongly agree” with the statement. One notable exception was noted with the implementation of conducting a holistic review of all applicants. More than three-fourths of “strongly agree” respondent program directors implemented this method compared to only half of “not strongly agree” respondents. A previous study conducted within an internal medicine residency program found that a holistic review of applicants increased the percentage of URM applications reviewed by 6.3%, increased URM interviews conducted by 8.5%, and resulted in an overall 19.2% increase in URM candidates matriculated . These fFellowship directors held fellowship programs most responsible for increasing diversity, followed by residency programs and medical schools. These responses suggest that directors of cardiology fellowship programs believe that increasing representation in cardiology requires the cooperation of administrators in undergraduate medical education as well as post-graduate training. Crowley et al. takes this argument a step further and encourages program directors to diversify the field through the development of a “deep pipeline” recruitment model through a partnership with local universities, high schools, and even elementary schools to recruit URMs to cardiology through early exposure to the field as a potential future career option . FurtherThis study adds to the literature in two ways: (1) It informs cardiology fellowship program directors of what many of their colleagues believe increases diversity in their programs as well as which strategies they implemented. These strategies may offer options for program directors seeking to increase diverse applicants. (2) These results may inform future research to identify which strategies statistically significantly increase the number of applicants and residents that are underrepresented minorities. In particular, prospective studies are needed with clearly defined metrics to track the success of initiatives to increase the number of URM applicants and eventual residents.LimitationsOur study is not without limitations. Despite not using a previously validated survey instrument, we employed a robust developmental process that included review by content experts, presentations of the survey tool at local conferences to invite methodological critiques, and appraisal by associate cardiology program directors. Although our response rate was 28.4%, we used several strategies to drive responses, such as sending reminder emails and offering an optional lottery incentive. Notably, although we received responses from programs from different community settings and geographic locations, we were unable to compensate for non-response bias in this study, in which participants who responded to the disseminated survey may have disproportionately been interested in achieving increased racial and ethnic diversity within their programs compared to non-respondents. Also, this study was not designed to investigate female representation in cardiology fellowships, although women remain underrepresented in cardiology sub-specialty training . TherefoThis cross-sectional study of cardiology fellowship program directors found that program directors (1) overwhelmingly supported increasing diversity in cardiology fellowship programs, (2) endorsed multiple strategies to increase diversity within their programs, and (3) believed that fellowship programs were most responsible for increasing diversity in the subspecialty of cardiology. Additionally, this study was able to quantify the amount of URM representation in ACGME accredited cardiology programs and identified that a majority of cardiology programs have URM faculty in leadership roles, and nearly a quarter of the surveyed program directors were URMs themselves. Interestingly, program directors held fellowship training programs most responsible for increasing URM representation in the field of cardiology. It is our hope that the results of this study may inform current cardiology fellowship program directors of which interventions are being used in other programs, which strategies are most supported by their peers, and which initiatives may yet need to be implemented. These findings may also be of value to medical students and resident physicians interested in applying to cardiology fellowships. Future research is needed to determine which strategies are most effective to increase URMs in cardiology fellowship programs in the United States.


Universal varicella vaccination might reduce opportunities for varicella-zoster virus (VZV) exposure and protective immunological boosting, thus increasing herpes zoster incidence in latently infected adults. We assessed humoral and cell-mediated immunity (CMI), as markers of VZV exposure, in adults aged ≥50 years.We repurposed data from placebo recipients in a large multinational clinical trial (ZOE-50). Countries were clustered based on their varicella vaccination program characteristics, as having high, moderate, or low VZV circulation. Anti-VZV antibody geometric mean concentrations, median frequencies of VZV-specific CD4 T cells, and percentages of individuals with increases in VZV-specific CD4 T-cell frequencies were compared across countries and clusters. Sensitivity analyses using a variable number of time points and different thresholds were performed for CMI data.P < .0001) but not by VZV circulation. No significant differences were identified in VZV-specific CMI between participants from 2 high versus 1 low circulation country. In 3/5 sensitivity analyses, increases in CMI were more frequent in high VZV circulation countries (.03 ≤ P < .05).VZV-specific humoral immunity from 17 countries  varied significantly between countries (We found no consistent evidence of reduced VZV exposure among older adults in countries with universal varicella vaccination.NCT01165177. By repurposing humoral and cell-mediated immunogenicity data from a large multinational trial enrolling adults aged ≥50 years, we found no consistent evidence of differences in varicella-zoster virus exposure between countries with widely circulating varicella versus countries with universal vaccination. Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects most individuals, usually during childhood, although its incidence may be delayed in warmer climates . PrimaryIn 1965, Hope-Simpson hypothesized that VZV reactivation is under an immunological control that is periodically boosted by exposures to varicella  and/or by prior subclinical VZV reactivation events  .Based on the observation that herpes zoster incidence was higher in adults living without children compared to those living with children (a surrogate for VZV exposure), Brisson et al  construcSeveral countries have implemented universal varicella vaccination , leadingFor this post hoc analysis, we repurposed data from a multinational randomized clinical herpes zoster vaccine trial  to deterBox 1.The varicella-zoster virus, which causes varicella in childhood, remains latent and may reactivate later in life, leading to herpes zoster disease.Protection against varicella-zoster virus reactivation and herpes zoster is expected to be boosted by 2 mechanisms: exposure to varicella (exogenous boosting) and/or by asymptomatic reactivation of the virus (endogenous boosting). To induce exogenous boosting, the varicella exposure should be intense enough to stimulate an immune response.Varicella vaccines are highly efficacious in preventing varicella disease in children and are routinely used in several countries. Many other countries have delayed implementation based on the hypothetical concern that decreased exposure to varicella (through vaccination) may lead to an increase of herpes zoster disease in older adults, due to the lack of exogenous boosting.We assessed the differences in varicella-zoster virus immune status among participants ≥ 50 years of age using samples from a clinical trail conducted in 18 countries. The objective was to seek evidence that there are more opportunities for intense varicella exposure in countries with endemic varicella (high virus circulation) compared to those with varicella vaccination programs (low virus circulation).We did not detect any differences in immunity among populations from different countries.In our analysis, we found no consistent evidence that varicella vaccination programs reduce opportunities for varicella-zoster virus exposure among older adults.The results should be carefully considered by national authorities when implementing vaccination policies.This post hoc exploratory analysis was based on data derived from placebo recipients in a large, placebo-controlled, randomized clinical trial of the adjuvanted recombinant zoster vaccine, conducted in 18 countries across North America, Latin America, Europe, Asia, and Australia, between August 2010 and July 2015   of 1067 and 119 [54.6%] of 218) and of Caucasian/European heritage (747 [70.0%] of 1067 and 128 [58.7%] of 218). Characteristics of the overall study population enrolled in the placebo arm of ZOE-50 have been previously published  versus 464.79 [SD 565.55]). A summary of CMI data is shown in IFN-γ+(+) T cells was not statistically significant (P = .56). Point estimates of the median frequency of VZV-specific CD4IFN-γ+(+) T cells/106 CD4 IFN-γ+(+) T cells counted were higher in the 2 countries with high circulation compared to the country with low circulation: 414  for Czech Republic and 448  for Japan versus 380  for the United States.CMI samples were available from 3 countries: 2 with high and 1 with low circulation. CMI status at month 0, assessed by the mean frequency of CD4P values below 5% were observed for the results of participants with data available for at least 2 time points (both for at least 2-fold and 3-fold increases) and all 5 time points (only for at least 3-fold increase). However, CIs were large and overlapping for all analyses.In the main analysis, based on participants with at least 2-fold increases and data available for the first 4 time points, no statistically significant difference was observed between countries with high and low VZV circulation . When seConcerns that universal varicella vaccination could result in increased herpes zoster incidence are based on the assumption that in varicella-endemic settings, large portions of the latently VZV-infected population experience periodic VZV exposure of sufficient intensity to stimulate the still unrecognized components of immunity that control reactivation . HoweverChanges in humoral immunity of adults following reexposure to VZV have been explored in several studies. Two studies , 23 founIFN-γ+(+) T cells of participants from countries with high versus low VZV circulation levels. This finding supports the hypothesis that VZV exposures in the general population are insufficient to elicit detectable immune responses in a significant proportion of individuals. Similar results were reported by Ogunjimi et al [IFN-γ+ T cells of grandparents with known reexposure to VZV over 1 year, or compared to controls.Age-related decreases of VZV-specific T-cell frequencies are associated with increased risks of herpes zoster, although no proven correlates of protection have been identified . Circulami et al , who fouThe kinetics of circulating VZV-specific CD4 T cells are somewhat similar during symptomatic reactivation and following reexposure to VZV, reaching a peak within the first 2 to 4 weeks and then declining by week 6 to modestly elevated and persistent levels , 23. In We observed transient CMI increases in all 3 countries with available data and although percentages were lower in the United States (a country with low VZV circulation), the differences were not statistically significant in the main analysis. In 3 of the 5 sensitivity analyses, differences between the United States and the 2 countries with high circulation (the Czech Republic and Japan) were marginally significant, although CIs were wide and overlapping for all analyses. While this could reflect reduced VZV exposure, it can also represent a country effect, considering that only 3 countries were included in this analysis. Although it is not clear whether endogenous boosting protects against development of herpes zoster , 28, it In certain settings and subpopulations, the protective effects of reexposure to varicella seem quite possible. For example, in adults, a protective effect of living with children was observed in the prevaccination era but disappeared in the years after vaccination implementation , and a mOur study has limitations. Humoral immune responses following VZV reexposure decline rapidly, with cellular immune responses being somewhat more durable ; the outIn conclusion, we assessed humoral and CMI to determine whether VZV exposures are more common in adults aged ≥50 years in countries with widely circulating varicella versus countries with universal vaccination and good varicella control but found no consistent differences. Our results suggest a credible explanation why countries that have introduced varicella vaccination programs have not experienced postulated increases in herpes zoster incidence and can inform national authorities considering implementation of universal varicella vaccination. Repurposed immunological data from completed clinical trials can provide useful information with which to address important questions relating to the dynamics of infectious diseases.The Journal of Infectious Diseases online. Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.Supplementary materials are available at jiab500_suppl_Supplementary_DataClick here for additional data file.jiab500_suppl_Supplementary_FigureClick here for additional data file.


C1orf185  and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene,  Infertility, or the inability to conceive, remains a prevalent and ongoing global health concern . InfertiA comprehensive and in-depth understanding of genetic factors in spermatogenesis abnormalities will play important roles in the clinical diagnosis, treatment, and genetic counseling of male infertility. Currently, the discovery of novel genetic factors in idiopathic infertility is a major challenge in the fields of reproductive medicine and andrology. The highest frequency of known genetic factors contributing to male infertility (25%) is in azoospermia . The diaThe use of whole exome sequencing (WES) for the detection of disease-causing variants of genetic diseases is a major clinical application of next generation sequencing (NGS). WES allowed the discovery of an increasing number of monogenic defects of NOA with a current list of 38 candidate genes . HoweverThe mouse model, due to its amenability and its genetic and physiological similarities with man, is extensively used to study human health and diseases. The creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Knockout (KO) mice have been pivotal in studying gene function in vivo. Historically, the study of KO animals is the “gold standard” to determine whether a gene’s function is essential in vivo. In the era of CRISPR/Cas9-based genome engineering, the generation of genetically modified mice becomes much more flexible, accurate, efficient, and cost-effective. The functions of testis-specific genes can be assessed in vivo by creating male mice carrying a null mutation in the genes of interest. Upon the production of KO mouse lines, the essentiality of the deleted genes can be readily determined by examining the fertility of homozygous male offspring.C1orf185 or CCT6B). To study the impact of loss-of-function (LoF) bi-allelic variants of these genes in sperm production and male fertility, we generated two KO mouse lines by introducing homozygous frameshift mutations in the corresponding orthologs 4930522H14Rik and Cct6b. Homozygous KO males were fertile and showed normal sperm production, thus excluding an essential role of these genes for spermatogenesis and male fertility. In the present study, we investigated by WES two unrelated infertile patients born from consanguineous parents and displaying idiopathic non-obstructive azoospermia associated with severe hypospermatogenesis. After variants filtering and prioritizing, we retained for each individual a homozygous truncating variant in a testis-specific gene . Informed consent was obtained from all individuals participating in the study according to local protocols and the principles of the Declaration of Helsinki. The study was approved by local ethics committees, and samples were then stored at the Fertithèque collection declared to the French Ministry of Health (DC-2015–2580) and the French Data Protection Authority (DR-2016–392).Genomic DNA was isolated from blood samples or saliva using the Oragen DNA Extraction Kit . Genetic data were obtained from Novogene . Coding regions and intron/exon boundaries were sequenced after enrichment using SureSelect Human All Exon V6 from Agilent.An alignment-ready GRCh38 reference genome , decoy, and human leukocyte antigens (HLA)) was produced using “run-gen-ref hs38DH” from Heng Li’s bwakit package . The exohttps://github.com/ntm/grexome-TIMC-Primary, accessed on 5 November 2018) takes FASTQ files as input and produces a single merged GVCF file, as follows. Adaptors are trimmed and low-quality reads are filtered with fastp 0.20.0 [The first module  takes this merged GVCF as input and produces annotated analysis ready TSV files. This is achieved by performing up to 15 streamlined tasks including the following: low-quality variant calls (read depth (DP) < 10, genotype quality for variant filtration (GQX) < 20, or less than 15% of reads supporting the ALT allele) are discarded; Variant Effect Predictor v92 [The second module . Analyses were performed using SeqScape software 3.0 . Primer sequences and their expected product sizes are summarized in 4930522H14Rik and Cct6b, respectively, as described in CrispR/Cas9 gene editing was used to knockout DNA for genotyping was isolated from tail biopsies. Tail biopsies (2 mm in length) were digested in 200 µL of Direct PCR Lysis Reagent (Tail)  and 0.2 mg of proteinase K for 12–15 h at 55 °C followed by 1 h at 85 °C for proteinase K in activation. The DNA was directly used for PCRs. PCR products were separated by 2% agarose gel electrophoresis. Genotypes were determined according to the migration pattern. Primers are described in All procedures were conducted in Geneva until the birth of the modified litters (F0 generation). Animals of interest were then transferred to the University Grenoble Alpes (UGA). All animal work was conducted according to the ethical guidelines of the French local Ethical Committee  and the Direction Générale de la Santé (DGS) for the State of Geneva.Mice were housed with unlimited access to food and water and were sacrificed by cervical dislocation after they were eight weeks old, which means that they were pubescent and that their reproductive organs were fully established. To test fertility, pubescent homozygous KO and WT males (8 week-old) were mated with WT females for at least two months. The females were checked for the presence of vaginal plugs and pregnancy. The number of mice achieving a pregnancy and the litter size of each mating set or pregnancy were recorded. We tested in this study (due to the COVID-19 pandemic) a single KO animal and at least two WT animals for each mouse line. Because the main aim of this study was to validate or invalidate the implication of the identified candidate genes in NOA, we focused our phenotypic analysis on quantitative spermatogenic defects in KO mice, leading to oligozoospermia or azoospermia. The qualitative defects such as morphological and functional defects of sperm cells produced by KO animals were not fully investigated in this study, and therefore related data are not reported in the present paper.To determine sperm concentration, adult males were euthanized and sperm samples were collected from the cauda epididymis and vas deferens. Spermatozoa were released in 1 mL of PBS solution by making a series of incisions in these ducts. Sperm number was determined using a hemocytometer under a light microscope, after 2 min of fixation by paraformaldehyde 4% to immobilize motile cells. Sperm motility and morphology were examined under a light microscope to rule out any obvious abnormalities compared to WT animals.To analyze testicular integrity, testes from adult wild-type (WT) and KO mice were fixed by immersion in 4% paraformaldehyde (PFA) for 14 h, embedded in paraffin, and sectioned (7 µm). For histological analysis, after being deparaffinized, slides were stained with hematoxylin and eosin. The colored sections were digitized at ×20 magnification through an axioscan slide scanner  equipped with a motorized X–Y-sensitive stage. t-Tests were used to compare the WT and KO samples. Data represent mean ± SEM or SD, as indicated. Statistical tests with a two-tailed p-value ≤ 0.05 were considered significant.n represents the number of biological replicates. For sperm analyses, for each replicate, more than 100 sperm were assessed per condition. Statistical analyses were performed with GraphPAD prism software 6 . Two unrelated men, P0280 and P0365, with an age of 44 and 43 years, respectively, sought medical care at the Clinique des Jasmins in Tunis (Tunisia) for primary infertility. Both patients were of North African origin and were born from related parents A. AnalysMen displaying NOA may have sperm cells in their testes that could be retrieved through a surgical procedure such as microdissection testicular sperm extraction (mTESE) . mTESE wC1orf185 or CCT6B. All the identified variants were rare  according to the gnomAD database and were absent from our local control cohort (n = 445). These candidate genes were predicted, according to in silico databases, to be highly and specifically expressed in testes contrasting with an unknown biological function. We also looked for other genes with another tissue expression pattern and did not find any relevant candidate variants. We paid particular attention to genes with high expression in both male and female gonads when analyzing the exome data from P0280. The familial history of this subject showed that he has two infertile sisters. This information could orientate us toward a common genetic cause that explains the infertility of these siblings, particularly if the women display primary ovarian insufficiency, a comparable phenotype with NOA in men. However, these cases have not been documented and in addition, we were unable to obtain their DNA samples to perform genetic segregation analyses.We used a WES-based strategy to investigate two patients in order to identify the potential genetic origin responsible for their infertility. Given the familial history of consanguinity, we postulated that these genetic factors had a likely autosomal recessive inheritance. Therefore, we focused our analyses on homozygous variants with an allelic frequency (AF) <1%. After variant filtering and prioritizing, we selected and retained for each patient a homozygous truncating variant in C1orf185 (chromosome 1 open reading frame 185) is located on chromosome 1 (1p32.3). The canonical transcript  contains five exons coding for a protein of 199 residues. We identified a nonsense variant  located in exon 3 and was suspected to either produce a truncated protein or to induce the degradation of the mRNA by the nonsense-mediated mRNA decay (NMD). CCT6B (chaperonin containing TCP1 subunit 6B) is located on chromosome 17 (17q12). The canonical transcript  contains 14 exons coding for a protein of 530 residues. We identified in this gene a splicing variant (c.615-2A>G) affecting the second nucleotide of the splice acceptor consensus sequence located in intron 5  from F0 generation with WT males to obtain heterozygous mice (F1 generation). F1 heterozygous males were then mated with heterozygous females carrying the same mutation to obtain homozygous mutated mice (F2 generation).We then designed for each gene two guide RNAs (gRNAs) targeting two coding sequences located in a single exon to produce a large intra-exonic frameshift deletion. The Cas9 protein and the dual-gRNAs were injected during ICSI procedure in mature oocytes. We then transferred embryos in pseudopregnant females and obtained 21 and 34 pups (F0 generation) after targeting Phenotypic analyses were carried out for the homozygous KO male mice in parallel with the same-aged WT controls from the F2 generation to investigate spermatic and testicular phenotypes. In addition, we tested the fertility of homozygous KO animals. No abnormal somatic development or behavior was observed in any of the KO mouse lines generated in this study.4930522H14Rik (Ortholog of C1orf185) KO line was generated using two gRNAs designed to target two coding sequences located in exon 2   . Histological analysis of testicular section from KO and WT animals showed normal architecture of the testicular parenchyma (The n exon 2 A. Genotyn exon 2 B. We seln exon 2 B. Homozy= 0.195) C. We per= 0.221) D. We col= 0.221) E  . Histological analysis of testicular sections from KO and WT animals showed that they presented similar parameters with normal parenchyma architecture and functional spermatogenesis (To obtain the n exon 4 A. We pern exon 4 B. A 155 n exon 4 B. Littern exon 4 C. Statis= 0.457) D. After = 0.457) E and  techniques and the availability of WES for research and clinical practice allowed for the discovery of several novel candidate genes responsible for spermatogenic defects and male infertility ,19,20. NC1orf185  and CCT6B (c.615-2A>G). To assess the implication of these candidate genes in NOA and male fertility, we invalidated their mouse orthologs using the CRSIPR/Cas9 system and generated two KO lines. The same strategy has previously allowed us to gain a better understanding of the genetic etiology of several spermatogenic defects such as NOA and monomorphic teratozoospermia [Despite the robustness of WES to study the genetic component of idiopathic NOA, many deleterious variants in genes with uncharacterized biological function are classified as variants of unknown clinical significance (VUS) . Althougospermia ,29,30.Using CRSIPR/Cas9 technology, gene edition in mice is now relatively easy and fast, allowing for the generation of several knockout and/or knockin lines in only a few months . The met−/−4930522H14Rik and −/−Cct6b males displayed normal fertility compared to WT males born from the same parents. We also showed that KO males from both lines presented normal sperm parameters and spermatogenesis compared to control littermates.In this study, we focused our phenotypic analyses to study the reproductive system and demonstrate whether adult male mice exhibited an altered spermatogenesis. We performed a fertility test by mating homozygous KO males with WT females and observing the litter size following at least two months. We observed that −/−Cct6b mice generated by the CRISPR/Cas9 system by targeting the same exon (4/14). Concordant with what we describe here, they found that males exhibited no differences in development, fertility, testis weight, sperm counts, total motility, and spermatogenesis relative to control littermates, thus confirming our results [In June 2021 and during the preparation of our manuscript, Yang et al. published an interesting paper reporting the reproductive phenotype of  results .During the past five years, many teams searching for target proteins for non-hormonal male contraceptive drugs published their studies reporting the dispensable role of numerous testis-enriched genes in spermatogenesis and male fertility through a massive production of KO mice using the CRISPR/Cas9 system ,34,35,36Whole exome sequencing (WES) is a robust and one of the most comprehensive genetic tests to identify rare disease causing variants and new candidate genes in a wide variety of spermatogenic defects associated with male infertility. Functional interpretation performed in candidate genes is now a critical step to validate their involvement in the studied phenotype. Among the available approaches nowadays, generation of transgenic animal models presents a powerful tool to study the function of candidate genes and their physiopathological implication. The recent development of the CRISPR/Cas9 system has made this process easier and faster, thus combining the use of WES to identify candidate genes and CRISPR/Cas9 to generate knockout mice is a very promising strategy to improve the genetic diagnosis yield and refine the interpretation of variants found in new candidate genes in male infertility.


We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future.Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point; we review cell type – specific expression pattern of the disease – causing genes and reflect on genetic and cellular treatment approaches including  Although each individual leukodystrophy is a rare disease, the collective overall incidence has been reported to range between 1: 5000 and 1:100.000 live births, rivaling the incidence of acquired white matter diseases including multiple sclerosis  can be very effective in attenuating disease progression. This has been the preferred treatment in pre-symptomatic X-linked Adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD) and Krabbe disease patients over the past 30 years . The ratex vivo and in vivo approaches, each using either viral or non-viral vectors for delivery of coding or non-coding therapeutic nucleic acids. Despite substantive progress in the field, which have expanded gene therapy strategies personalized approaches dependent on the patient, the specific disease, and its progression at treatment are key precepts.In the following section we provide a perspective on recent progress in the field. Broadly, gene therapy approaches can be divided into Ex vivo gene therapy builds on HSCT and largely overlaps with cell therapy in that patients’ own stem cells are extracted genetically modified via chromosomal integration of a therapeutic DNA, expanded, analyzed and reintroduced into the same patient. The advantage of this approach is that graft versus host disease and graft failure as well as other immune complications are largely eliminated  cDNA in the STARBEAM study (ALD-102) in X-ALD patients (ARSA) HSC-GT for early onset MLD has recently received a marketing authorization by the European Medicines Agency (EMA) on the basis that the benefits outweigh the risks, becoming the third approved HSC-GT after ZyntegloTM and StrimvelisTM  via lentivirus mediated gene addition and TALEN mediated gene editing. Direct injection of either of these genetically modified induced pluripotent cells (iPCs) into a CD mice resulted in pronounced histological, pathophysiology and motor behavior improvement  has taken the center stage in the development of gene therapies for neurological disorders (® (Voretigene neparvovec) has been developed for Leber’s congenital amaurosis using subretinal injection and Zolgensma® (Onasemnogene abeparvovec) is directed at spinal muscular atrophy in children under the age of two years, utilizing intravenous (IV) delivery.isorders . Of the In vivo gene therapy for leukodystrophies requires therapeutic gene transfer to the CNS which is compromised by the skull, the blood – brain barrier (BBB) and cerebrospinal fluid (CSF) – brain barrier (CBB). Delivery methods for rAAV to the brain include direct intraparenchymal (IP) injection, intracerebroventricular (ICV), lumbar intrathecal (IT) intra cisterna magna targeted intrathecal (ICM) and IV injections as well as nasal delivery which have been addressed in excellent recent reviews  DNA genome. Cell surface receptor binding, internalization, endosome escape and trafficking as well as aspects relating to immune escape and AAV production including stability and assembly are mediated through the capsid. As such the AAV capsid is the principal determinant of biodistribution, cell and tissue tropism and the site of interference with circulating neutralizing antibodies. The AAV capsid is a 60mer composed of three viral proteins VP1, VP2 and VP3 encoded in a single open reading frame, that assemble in a 1:1:10 ratio. Based on sero – reactivity, at least 13 human and non-human primate (NHP) AAV serotypes have been isolated, but seminal work from Guangping Gao and James Wilson’s lab identified more than 100 variants in NHPs in the early 2000’s and divided these naturally occurring AAVs into phylogenetic clades on the basis of functional and serologic similarities , 2005. SComprehensive investigations of AAV tropism have been performed for select AAV serotypes and variants. Intraparenchymal AAV injection into the adult rodent brain using constitutive promoters to drive transgene expression suggests that most AAVs preferentially transduce neurons. Amongst others these include AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39 and AAV.cy5 with superior transduction efficiencies achieved by AAV1, AAV9, AAV.rh10. . While t+ HSC of which AAVHSC7, AAVHSC715 and AAVHSC717 effectively crossed the NHP BBB and transduce astrocytes, oligodendrocytes, neurons and cells of the choroid plexus  into the AAV capsid to elicit a desired receptor binding function . Developcis-acting element (CRE) required for rAAV vector production, the entire wt genome is replaced by the recombinant expression cassette consisting of promoter, genetic payload and regulatory elements. The ITRs harbor the essential replication and packaging signals and are vital for episome formation and protection from cellular nucleases. Most AAV vectors are produced via cross-packaging where ITRs and Rep genes from one serotype are packaged into the capsid from another. The most commonly used ITRs stem from AAV2 (The key component of a gene therapy is the genetic payload and the regulation of its expression. The wildtype (wt) ssDNA genome of naturally occurring AAVs spans approximately 4.7 kilobases (kb) and, restricted by virion size, packaging capacity is capped at ∼5 kb or 2.3 kb in self-complementary (sc) genomes . scAAV grom AAV2 .Ideally a gene therapy should aim to restore as close to natural gene expression profile as possible to reset the proteomic and metabolomic micro-environment. Genetic payloads and mechanisms to control transgene expression have become increasingly versatile and can include complete coding sequences for proteins, untranslated regions (UTRs) and regulatory elements beyond the polyadenylation signals including post transcriptional regulatory elements, ribozymes, aptamers, regulatory RNAs like short hairpin RNAs (shRNAs), microRNAs (miRNAs), other non-coding RNAs and guide RNAs for gene editing . These ccis-regulatory elements (CREs) carrying clusters of binding sites for silencers and enhancers may be located up to 1 megabase pair up or downstream in intergenic or intronic regions. rAAV – mediated transgene expression has predominantly employed strong constitutive eukaryotic, viral or hybrid promoters that are thought to drive expression in all cell types. Prominent examples include the cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PGK), elongation factor 1 α (EF1α), ubiquitin C (UbiC) and chicken β-actin promoter and various hybrids thereof. The 1.6kb CAG promoter comprises the CMV immediate early enhancer and the chicken β-actin promoter fused to the first exon and intron and the splice acceptor of the rabbit β-globin gene. In light of the limited packaging capacity of AAV vectors, size reduction of the of CAG promoter hybrids to ∼ 800 bp by replacing the 5’-UTR of CBA with either a truncated simian virus 40 intron or minute virus of mice VP intron were welcomed achievements (The first level of gene regulation is governed by the promoter that recruits transcription factors (TFs) in a sequence specific manner to initiate transcription through RNA polymerases. The proximal core promoter is generally located within the first 1 kb of the transcription start site, but additional evements . CBA hybevements . Direct 2+/calmodulin kinase II (CamKII) promoter successfully to drive AAV – mediated transgene expression in neurons. Shorter neuron specific promoters including the 0.47 kb human synapsin (hSYN1) promoter, a compacted 0.4 kb truncated neuronal-specific CamKII promoter and the 0.23 kb methyl CpG binding protein (MeCP2) promoter have been generated since  required for silencing activity in neurons, this promoter has since been compacted to a twofold more active 681 bp gfaABC1D promoter with comparable astrocyte specificity (3) variant targeting GFAP positive astrocytes in mouse basal ganglia. More recently, following the identification of aldehyde dehydrogenase 1 like protein 1 (ALDH1L1) as a pan-astrocytic marker, the 1.3 kb human ALDH1L1 promoter has restricted AAV – mediated transgene expression to astrocytes in certain brain regions in mice  promoter was the first to show clear astrocyte specificity in the AAV setting . Followicificity  and a gf in mice .in vivo (Oligodendrocyte specific expression in the context of AAV was first achieved with the 1.9 kb mouse myelin basic protein promoter (Mbp) promoter, but heavily depends on the timepoint of AAV infusion after onset of myelination in mice . This prin vivo . More rein vivo .In the context of targeting microglia, several promoters including CD68 and CD11b have been assessed with the F4/80 promoter being the most promising to date . Howeverin vivo , UTG8 (Ple267) and Olig1  . Many CRin vivo . This fuin vivo . Togethe–/–; Rag2–/–; Il2rg–/–Fah (FRG) mice with primary human hepatocytes where screening for functional transduction revealed that bioengineered capsids outperform natural serotypes  IV delivery of AAV vector (AAV8-MTM1) in the ASPIRO phase I/II clinical trial targeting X-linked myotubular myopathy (NCT03199469), is a reminder that despite comprehensive pre-clinical safety and efficacy studies and the excellent safety profile of AAV even at high doses, outcomes in first human trials may result in the severest adverse events (14 vg/kg) and genotoxic integration potentially increasing the risk of liver cancer founded on clonal hepatocyte expansion observed in dogs  emphasize the importance of continued pursuit of the safest and most efficient AAV vectors, particularly for the treatment of leukodystrophies in which the entire CNS needs to be targeted, which inherently requires a high vector dose , where this role is taken by Schwann cells . ParticuPLP1) gene duplication (Pelizaeus-Merzbacher disease (PMD) is a X-linked, recessive dysmyelinating leukodystrophy with the most common and severe forms caused by proteolipid protein 1  mediated toxicity. Direct intraparenchymal injection of scAAV1/2 mediating oligodendrocyte restricted expression of a synthetic miR-155 harboring a PLP1 directed short hairpin prevented oligodendrocyte demise, restored myelin, and improved neurological phenotypes and survival , particularly when packaged into oligodendrogliotrophic exosomes that effectively cross the BBB are another developing technology that may hold promise in future. Indeed, therapeutic promise has recently been demonstrated in the ‘jimpy’ mouse model of PMD where single administration of a lifespan .GJC2) gene  is a hypomyelinating leukodystrophy with very similar clinical manifestation to PMD but caused by autosomal recessive loss-of-function mutations in the gap junction protein connexin 47 (C2) gene . High leC2) gene . AAV1/2  therapy . Given tstration , 2018a,bTUBB4a miRNA. To prevent potential undesired effects associated with loss TUBB4A, targeting the miRNA to the untranslated region and simultaneous expression of transgenic wt TUBB4a both driven by a predominantly oligodendrocyte – specific promoter may be viable. The AAV serotype chosen, and associated delivery route, would need to efficiently transduce oligodendrocytes. Similar to the above, AAV – CRISPR mediated gene correction and potentially ASO may become additional treatment options in future.Typically, H-ABC presents in early childhood with varied rate of progression and severity involving complex pathology including spasticity, dystonia, dyskinesia ataxia and tremor. Motor function is commonly more severely affected than cognition. H-ABC has been associated with toxic ‘gain of function’ mutations in the TUBB4a gene encoding the microtubule associated protein tubulin β-4a . ResolutSOX10 gene replacement in oligodendroglial progenitor cells (OPC). The infantile forms may be particularly amendable to AAV – mediated gene replacement, but suitable capsids enabling selective OPC transduction following efficient delivery to the CNS across the CBB or BBB and appropriate promoters remain to be identified. Potentially AAV variants SCH9 or AAV.r3.45 that have been shown to effectively transduce NPCs are promising candidates.SOX10-associated peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease (PCWH) presents with a complex central and peripheral pathology . BecauseASPA) encoding gene. Its substrate N-acetylaspartate (NAA) is the second most abundant brain metabolite, being predominantly synthesized by aspartate-N-acetyltransferase NAT8L in neuronal mitochondria. Although the exact function of NAA remains enigmatic, lack of NAA hydrolysis by ASPA which is exclusively expressed in oligodendrocytes causes its toxic accumulation and severe white matter vacuolization. Age of onset generally correlates with disease severity and in the most common infantile form children present with hypotonia, macrocephaly and fail to reach developmental milestones in months after birth before progressively losing motor function, seizure development and premature demise 20 targeted predominantly neurons and achieved long-lasting therapeutic benefit by lowering NAA levels, preventing spongiform white matter vacuolization and markedly improved motor performance  gene rendering the lysosomal hydrolytic enzyme that is required for myelin turnover inactive. This leads to toxic accumulation of psychosine, a byproduct of galactosylceramide synthesis and GALC substrate  prior to disease onset in a naturally occurring canine model for Krabbe disease decreased psychosine concentration and inflammation, improved myelination, nerve conduction and extended lifespan from 16 weeks to at least 2.5 years are very promising (GALC at a lower dose (3.8x1013 vg) while ameliorating CNS and PNS disease only doubled the lifespan (14 vg/kg) with bone marrow transplant between P10 – P11 in twitcher mice extended median survival over 500 days  and delivery routes  have been trialed in mouse models of the disease, many lagging behind expectations. The severity of the disease and extent of gene replacement required to treat Krabbe disease is underscored by the fact that even in combination with bone marrow transplant it required neonatal IV, IT and intraparenchymal AAV9-CAGGS-al signs . Consideromising . In contlifespan . Recent 500 days . Althoug500 days . In a se500 days .GalC ablation appears to be highest between P4 and P6 before onset of myelination, and GalC expression is highest in OPCs, newly formed oligodendrocytes and astrocytes, but drops in mature oligodendrocytes . In its severe infantile presentation AxD is characterized by megalocephaly, hypomyelination, developmental delay, psychomotor decline and seizures culminating in premature demise (Alexander disease (AxD) is caused by autosomal dominant e demise . The brae demise .GFAP mRNA, simultaneous expression of transgenic wt GFAP is feasible. In a different approach, a single intrathecal injection of a GFAP mRNA targeted ASO with enhanced nuclease resistance, binding affinity and lower toxicity has achieved remarkable efficacy providing elimination of GFAP throughout the CNS, reversal of Rosenthal fibers and rescue of hippocampal neurogenesis in an AxD mouse model 1, or dominant heterozygous mutations in GLIALCAM disrupt membrane localization of MLC1 to astrocyte endfeet and astrocyte junctions, where it stabilizes water channels including aquaporin-4 and the inwardly rectifying potassium channel Kir4.1  related vacuolating leukoencephalopathy and the Oculodentodigital dysplasia with cerebral white matter abnormalities (ODDD), a hypomyelinating leukodystrophy caused by an autosomal dominant mutation in the Gap junction α 1 (Connexin-43) encoding GJA1 gene  associated ‘’hereditary diffuse neuroaxonal leukoencephalopathy with axonal spheroids’ and the ‘pigmentary orthochromatic leukodystrophy’  as well  protein . These m protein . This meInterestingly, RNAseq data assessing cell type – specific gene expression profiles in mice  demonstrEx vivo HSC-GT has shown immense promise halting disease progression when performed in pre-symptomatic or early symptomatic patients and is currently in various stages of clinical trial for lysosomal and peroxisomal storage disorders including MLD and X-ALD disease, (ARSA) deficiencies triggering an accumulation of the myelin lipid sulfatide in oligodendrocytes and Schwann cells, as well as several peripheral organs, because the enzyme can no longer support its degradation and recycling. In this most common and severe late-infantile form of the disease, children lose the ability to walk and talk within months, and following rapid progression of motor symptoms, commonly die within seven years of diagnosis making the success of ‘Libmeldy’ a truly remarkable testament to the field. Pioneering the technology, HSC-GT successfully halts disease progression in X-ALD, even though in this case the peroxisomal very long chain fatty acid transporter ABCD1 is not secreted. In the STARBEAM study, HSC-GT for X-ALD is now also in late-stage Phase II/III clinical trial . Recent success in early symptomatic treatment of adult-onset X-ALD using HSCT underscores the potential feasibility of this approach for adult-onset LSDs and the above mentioned microgliopathies  gene, Sandhoff disease associated with HEXB mutations and Sanfilippo syndrome, a mucopolysaccharidosis (MPSIII) stemming from deficiencies in α-N-acetylglucosaminidase (NAGLU) or N-sulfoglucosamine sulfohydrolase (SGSH) gene. Recent development of a bicistronic lentivirus HEXA and HEXB encoding vector promoting stoichiometric synthesis of both subunits of the enzyme is capable of targeting both Tay-Sachs and Sandhoff disease in HSC-GT  and systemic delivery of scAAV9 (NCT02716246) are investigated. A recently initiated phase I dose escalation study for Tay – Sachs and Sandhoff disease (NCT04669535) combines dual ICM and IT administration of AAV.rh8-HexA and AAV.rh8-HexB at a 1:1 with bilateral intrathalamic delivery.in vivo. However, the success of HSC-GT in combination with the observation that microglial damage precedes myelin damage indicate that development of therapeutic AAV vectors with better microglial tropism may promote therapeutic outcomes even for leukodystrophies like MLD that may require a broader cell type overlapping approach.Looking forward, bicistronic HexA and HexB which can circumvent dual AAV delivery have shown promise in murine models of Sandhoff disease . To dateDARS1)  underlie progressive ‘leukoencephalopathy with ovarian failure and epileptic encephalopathy 29’ (EARS1) cause ‘leukoencephalopathy with thalamus and brainstem involvement and high lactate’ (LTBL) (RARS1) cause ‘hypomyelinating Leukodystrophy 9’ (POLR3A and POLR3B) encoding genes and is typically characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism. Age of onset varies but averages four years of age and POL3A mutations are associated with more severe disease. POLR3A and POLR3B are the main building blocks of this RNA polymerase that transcribes tRNAs and ribosomal RNA (rRNA)  related leukodystrophies, ‘hypomyelination with brainstem and spinal cord involvement and leg spasticity’ (HBSL) caused by mutations in the cytosolic aspartyl-tRNA synthetase 1 (DARS1) , 2018, ‘utations , the cytathy 29’ , mutatio’ (LTBL) , arginylrophy 9’  and the rophy 9’ . SimilarA (rRNA) . To dateRNASEH2A, RNASEH2B RNASEH2C. AGS manifests as microcephaly with progressive spasticity, psychomotor retardation. Neonatal onset has been associated with an approximately 35% fatality in early childhood. RNASEH2 is a DNA repair enzyme and its absence leads to DNA damage response and accumulation of cytosolic DNA aggregates triggering inflammation. While expressed across neural cell types, astrocytes rather than infiltrating leukocytes were found responsible for high cytokine and particularly interferon-α production in post-mortem brains indicating that gene replacement therapies, while targeting all cell types, should first and foremost ensure good astroglial coverage, but not prevent expression in other cells. Either way, a gene therapy for RNASEH2 related leukodystrophies must be carefully designed and tested because the enzyme consists of three albeit small subunits and to provide constraints around stoichiometry will be challenging. In addition, it should be noted that RNASEH2C is considered a metastasis susceptibility gene and interfering with the expression of cell cycle regulated genes must be very carefully considered as it may increase downstream risks of malignancies (Aicardi-Goutieres Syndrome (AGS) is caused by mutations in a number of genes involved in nucleotide metabolism. These include gnancies .GLB1 that is expressed by all cell types in the CNS  characterized by the accumulation of intermediate metabolites that are frequently toxic at higher concentrations and prevent homeostasis, eventually triggering cell death. Many LSDs have significant neurological and CNS involvement. While cross-correction by externally delivered lysosomal enzymes can mitigate peripheral aspects of the pathology, in the CNS uptake is largely prevented by the BBB, promoting the development of gene therapies that have the potential to overcome limitations of traditional enzyme replacement therapies. We apologize to our colleagues in this field whose valued contributions could not be included in this manuscript due to space constraints. Recent reviews have provided comprehensive overviews on clinical progress of gene therapies . Aside f the CNS . Reflect the CNS . In cont the CNS , intrave the CNS . MSD pat the CNS . In LSDsformance . In futuEIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5) encoding genes, that show a clear genotype – phenotype correlation. The nucleotide exchange factor eIF2B is required for eIF2 delivery of initiator Met-tRNA to the ribosome and initiation of translation (VWM disease, also known as ‘childhood ataxia with central nervous system hypomyelination’ is a prominent leukodystrophy that is caused by loss of function in eukaryotic translation initiation factor 2B subunit 1 to 5 (nslation . Classicnslation . Based onslation  but the EIF2B genes are expressed at high levels throughout peripheral organs and peripheral phenotypes such as endocrine effects have been observed and may also need to be addressed. Nevertheless, the pre-clinical benefits achieved through glial progenitor cell therapy are encouraging. Since most AAV vectors that penetrate the BBB or CBB transduce astrocytes with high efficacy following IV or ICM administration, AAV gene replacement with constitutively active promoters appears to be a viable treatment option for severe infantile VWM disease. For treatment development, capsid and promoter selection would certainly benefit from a screening for ‘functional transduction’ in a humanized animal model.Recent evidence from a glial progenitor cell therapy study suggested that phenotypic improvement is correlated with increased astroglial differentiation underscoring the hypothesis that astrocytes are central to the VWM pathology . In addiex vivo gene therapies as well as AAV – mediated in vivo gene therapies have been approved by the FDA. In addition, the first ASOs have also successfully advanced to this stage. In addition, most big pharma companies are supporting development and increasingly managing to address issues associated with large scale production. Nevertheless, despite the immense potential and undoubtedly growing importance of these evolving genetic drugs in modern medicine, recent clinical and pre-clinical studies also expose risks and pitfalls underscoring the importance of a cautious and considered advance. In the case of ex vivo HSC-GT, this has ensured development of self-inactivating lentiviral vectors and rigorous mapping of integration sites. With regards to AAV – mediated in vivo gene therapy programs, which are establishing excellent safety and efficiency profiles, the recent tragic deaths of three patients in the high dose cohort of the ASPIRO trial targeting myotubular myopathy as well as preclinical evidence of toxicity and severe immune response associated with high vector doses following systemic AAV delivery are a reminder of the risks and imperative for refinement, particularly around vector dose. This is of particularly relevant to treatments for leukodystrophies and leukoencephalopathies that rely on relatively high vector doses associated with targeting the entire CNS. Since direct intraparenchymal injections are associated with risk of hemorrhage and infection and it has been estimated that about 100 injection sites may be required to achieve full CNS coverage, the discovery of BBB and CBB traversing AAV serotypes has moved systemic IV and CSF vector administration into focus. Importantly, comparison of delivery routes and modes of rAAV vector administration is ongoing, but recent studies indicate when administered into the CSF a substantially better vector spread throughout the CNS and spinal cord is achieved by injection into the cisterna magna over other cerebroventricular and intrathecal approaches. A key to dose reduction is to increase this ability in the next generation of AAV vectors while ensuring immune – evasion and substantial reduction in off-target tissue transduction. Among others, notable preclinical progress has been made in this field through discovery of novel naturally – occurring AAV variants, alongside rational capsid design, capsid shuffling peptide insertion combined with high throughput screening and targeted selection to extend the repertoire of vectors and associated cell and tissue targeting profiles.Nucleic acid-based treatments of neurological disorders have rapidly expanded in the last decade and the long – recognized potential for gene therapy is now becoming a reality for patients and families suffering from a multitude of devastating genetic diseases. The first hematopoietic stem cell-based cis-regulatory elements to achieve gene expression in the target cell we compiled current capsid and promoters that may be considered as a starting point with regards to development of improved targeting strategies and potentially toggleable expression control. Notably, recent findings that direct capsid – promoter interactions determine the cell type – selective gene expression in the CNS highlight the importance of screening these aspects in conjunction. Because beyond uptake, capsid components influence intracellular transport, stability and gene expression, and cross species differences are evident even when targeting the same cell type, it will be increasingly important to confirm functional transduction in humanized CNS models. AAV capsid / promoter combinations identified via screens resulting from selective pressure for functional transduction of the target cell in humanized models may be a promising strategy to achieve the needed vector dose reduction required for treatment of CNS disorders. Beyond vector engineering and choice, other patient specific, parameters in addition to body weight may need to be considered when choosing vector dose. These include developmental stage, disease progression, immunity, and metabolic profile. As gene therapies for leukodystrophies and leukoencephalopathies advance, the programs will need to broaden to assess and mitigate risk. In many, if not most cases, a single shot ‘Magic Bullet’ curative treatment may be elusive, but such challenges are driving the rapid advance in sophistication of the vectors and therapeutic payloads to yield genetic medicines to treat these devastating neurological diseases.By reviewing the natural expression profile of genes associated with leukodystrophies across neural cell types, we hope to provide a better understanding of which cells may naturally be important targets. Highlighting the importance of the choice of promoters and GJ led the project and wrote the draft manuscript. GJ, CR, and GH contributed to the manuscript preparation. All authors read and approved the final manuscript.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.


TGF-β/Smad signalling has been the subject of extensive research due to its role in the cell cycle and carcinogenesis. Modifications to the TGF-β/Smad signalling pathway have been found to produce disparate effects on neurogenesis. We review the current research on canonical and non-canonical TGF-β/Smad signalling pathways and their functions in neurogenesis. We also examine the observed role of neurogenesis in neuropsychiatric disorders and the relationship between TGF-β/Smad signalling and neurogenesis in response to stressors. Overlapping mechanisms of cell proliferation, neurogenesis, and the development of mood disorders in response to stressors suggest that TGF-β/Smad signalling is an important regulator of stress response and is implicated in the behavioural outcomes of mood disorders. The transforming growth factor-β (TGF-β) pathway consists of many genes involved in cell growth, differentiation, migration, and apoptosis ,2. TGF-βE. coli used to induce an immune response [Neuroplasticity broadly refers to the ability of the nervous system to responds to internal and external stimuli by reorganising its structure and function at the molecular, cellular, and organisational levels. These adaptations can be beneficial or harmful to the organism; consequently, neuropsychiatric disorders have been examined as a manifestation of deleterious neuroplasticity. Indeed, neuropsychiatric disorders have been characterised according to alterations in the limbic, fronto-striatal, and prefrontal circuits, which in turn generate disturbances in behaviour, cognition, and motivation . These bresponse . Neuroinresponse . Althougresponse , such asresponse ,19.Evidence from previous work suggests an association between TGF-β signalling and the phenomena of neuroplasticity and neurogenesis, as well as their combined effects on mood disorders ,21,22. BNon-canonical TGF-β signalling refers to molecular events that occur independent of the SMAD pathway. TGF-β activated kinase 1 (TAK1) is an important effector in non-canonical TGF-β signalling. TAK1 initiates a signalling cascade that activates c-Jun N-terminal Kinases (JNK) and p38/mitogen activated protein kinase (MAPK) in response to TNF/TGF-β signalling, as well as activates the IkB Kinase (IKK) and Nuclear Factor-κB (NF-κB) pathways. TAK1 is an important regulator of innate and adaptive immune responses , and is The MAPK signalling pathway has been shown to be involved in neuronal differentiation and axon growth . The MAPThe c-AMP response element binding protein (CREB) is involved in the TGF-β pathway, as TGF-β was shown to activate extracellular elements that activate CREB . TGF-β wTGF-β has been shown to play a role in inflammation and promote cell survival, induce apoptosis, and initiate proliferation and differentiation ,42. In mDespite extensive research on the effect of TGF-β on neurogenesis , the relBesides interacting with TGF-β, Smad3 also interacts with a host of other signalling molecules such as its close analogue Smad2 . AlthougTGF-β binds to the type II receptor that in turn forms a complex that phosphorylates Smad3. TGF-β receptors were found to be expressed on new cells in the neurogenic region of the dentate gyrus and the subventricular zone . SimilarSmad signalling has been implicated in epigenetic functions such as Jumonji domain-containing protein D3 (JMJD3). Knockdown studies showed that spinal cord  and retiGrowth arrest and DNA-damage inducible protein 45 (Gadd45) is another epigenetic regulator that is affected by the TGF-β signalling pathway, as shown by blocking of TGF-β and TrkB leading to the overexpression of Gadd45 in vitro . Gadd45 Neurogenesis is hypothesised to play a role in depression via changes to the rate of neurogenesis, specifically the deleterious effects from stress-related neurogenesis in the dentate gyrus. Neurogenesis in the dentate gyrus can impact behavioural output and the efficacy of antidepressant treatments. New neurones heavily affects hippocampal circuitry and behaviour. Clinical evidence shows hippocampal shrinkage is linked to depression. In the pre-clinical stage, depressive behaviour results in a host of adverse effects on hippocampal neurogenesis, including reduced proliferation of neural stem cells and reduced neuronal survival. Furthermore, antidepressant treatment in rats induced hippocampal neurogenesis over time, in a pattern that mirrors the delay in antidepressant efficacy in human subjects . One couAlthough a direct causal link between hippocampal neurogenesis and mood disorders seems unlikely, neurogenesis is nonetheless an important factor in the development of mood disorders. The neurogenic interactome model attempts to reconcile these opposing findings by taking into account various factors affecting neurogenesis. The neurogenic interactome considers the interactions between brain structures, the various functions of the hippocampus, and the heterogeneity of elements in the neurogenic niche . For exaThe pathogenesis of mood disorders has been hypothesised to be related to neuroplastic changes, particularly neuroimmune processes such as neuroinflammation, which might affect the central and periphery nervous systems to impact the neurobiology of depression. Neuroinflammatory processes are suspected to exert deleterious effects on various pathways, such as the hypothalamic-pituitary-adrenal (HPA) axis, neurogenesis, neuroimmune response, and neurocircuitry by upregulating apoptotic functions. Various mechanisms have been put forward on how the immune system regulates neurogenesis and neuroplasticity, particularly the role of T-reg cells on enhancing hippocampal neurogenesis via upregulating glial cell-derived neurotrophic factors and TGF-β. It was shown that T-reg cells are attenuated during depression, whereas T-reg cells are increased with antidepressant treatments. However, behavioural studies in animals with suppressed neurogenesis but not exposed to stressors showed that neurogenesis by itself does not produce a depressive phenotype, as there were no differences in the behaviour between animals with ablated neurogenesis and controls .Stress has been shown to induce anxiety and depressive disorders by disturbing the HPA axis-regulated release of glucocorticoids ,78. In pA connection between stress and neurogenesis has also been observed, in that a reduction of neurogenesis precipitated the development of stress-related disorders such as post-traumatic stress disorder . This coThe hippocampus has been implicated in the development of depressive disorders. Neuroimaging studies and post-mortem examinations showed an association between reduced hippocampal volume and the duration of depression ,94,95. HThe amygdala has also been implicated in the pathogenesis of anxiety disorders, likely caused by hyperexcitability of neurones in the amygdala ,99. FurtThe TGF-β pathway has been implicated in a wide range of processes. This is evident by reports showing increased TGF-β in circulation predisposes an individual to hypertension  and polyTGF-β plays an important role in the central nervous system as a response to injury and trauma and functions as a signal to initiate cellular repair and protection ,115. TGFThe potential for TGF-β to be applied in a therapeutic context has been considered in the context of oncology where anti-TGF-β treatment has been proposed to target tumour cells and microenvironment . This coAlthough there is a link between neurogenesis and depression, several studies ,129,130 Analysis of human hippocampal neurogenesis showed that neurogenesis takes place at a roughly equivalent rate to that in middle-age rodents  and persThe observation that the inhibition of neurogenesis does not replicate the stress-induced hippocampal volume loss or behavioural changes indicates that neurogenesis is not the sole mechanism in the manifestation of depressive behaviours, and it is likely that stress affects other contributory factors. Regarding the loss of neurogenesis, previous work found it could be induced by autophagic cell death of neural stem cells. It was shown that deletion of Atg7, an important molecule in the formation of autophagosomes, resulted in neuroprotective effects in chronic restraint stress (CRS) and cortisol injection . This maIn conclusion, the TGF-β pathway is involved in many functions and is required for embryonic viability; however, its role in neurogenic mechanisms and their implication on mood regulation are largely unknown. Based on the available evidence, the TGF-β pathway potentially exerts effects on neurogenesis via the canonical pathway. These effects may in turn play a role in the development of mood disorders such as depression and pathological anxiety. Inactivation of Smad3 in animal models of depression induced by chronic restraint stress reveals TGF-β/Smad3 signalling together with non-canonical pathway components such as TAK1 and Erk play potential roles in hippocampal neurogenesis.