Historically, transitioning gender was seen as precluding transgender people from having children in the future. However, there are now increased reproductive options available to transgender people, with such options also available to non-binary people . These options include undertaking fertility preservation if genetic children may be desired in the future. Despite these increased options, there is still only a limited amount of international research exploring the views of transgender and non-binary people on fertility preservation.This mixed-methods study draws on a convenience sample of Australian transgender and non-binary adults, focused on their decision making about fertility preservation. The questionnaire was constructed by the authors, drawing on previous research. Participants were recruited via Australian organisations and groups made up of and/or working with people who are transgender or non-binary. The questionnaire was open from January–February 2018. The final sample included 409 participants. Statistical analyses were conducted on the closed-ended responses. Open-ended responses were analysed via a conventional content analysis.Decisions about fertility preservation were influenced by views on the importance of genetic relatedness, willingness to delay transition, economic resources, already having children or desiring children in the future, and the views of significant others. Advice or counselling prior to decision making was received only by a minority of participants. Very few participants (7%) had undertaken fertility preservation, although 95% said that fertility preservation should be offered to all transgender and non-binary people. Participants who viewed genetic relatedness as important were more likely to have undertaken fertility preservation.The findings indicate that fertility preservation should be made available as an option to all transgender or non-binary people prior to undertaking treatment which may impact on fertility. However, it should also be recognised that not all people who are transgender or non-binary will want to undertake fertility preservation, and that not all people may be able to afford to. In the past, transgender people were seen as automatically excluded from having children. However, the reproductive rights of transgender people have been increasingly recognised, as too have those of non-binary people . This may include preserving fertility should transgender or non-binary people wish to have children in the future. Yet there is little research which explores the views of transgender and non-binary people on fertility preservation.To explore such views, a questionnaire was designed by the authors. The questionnaire was completed by 409 transgender and non-binary adults in Australia.The findings from the questionnaire show that the people who completed the survey made decisions about fertility preservation depending on several reasons. The reasons included how important it was to be genetically related to a child, how willing they were to wait to transition gender, how much money they had, whether they had children already or if they wanted to have children in the future, and the views of people around them. Only a small number of people who completed the survey had preserved their fertility. Nearly all said that fertility preservation should be offered to all transgender and non-binary people.The findings suggest that the option for preserving fertility should be available to all people who are transgender or non-binary. However, not all transgender or non-binary people will want to preserve their fertility, or will be able to afford it.Standards of Care for the Health of Transsexual, Transgender, and Gender Nonconforming People (SOC), however, in their seventh version [Historically, transitioning gender was seen as precluding transgender people from having children in the future, with sterilisation often being mandatory, or treated as axiomatic in the context of gender affirming surgery. As such, whilst some transgender people may have had children prior to transitioning, the potential for future children was typically not considered. The World Professional Association for Transgender Health (WPATH)  version , introdu version . People  version  Importan version  Perhaps The country in which transgender or non-binary people live constitutes a formative context in terms of experiences of fertility preservation. Therefore, it is important to provide a brief background to the Australian context as it is the setting for the study reported in this paper. There are no specific laws or policies banning transgender or non-binary people from undertaking fertility preservation or assisted reproductive technology in Australia, although in some jurisdictions legislation enables providers to object on religious grounds. However, fertility preservation is expensive and is only covered by the Australian public healthcare system if it is classified as being medically necessary. Whilst for oncology patients fertility preservation is seen as medically necessary, for transgender and non-binary people it is not. Estimates from 2010 suggest that, depending on the clinic, sperm banking costs $300–$400 a year in storage fees, sperm aspiration costs $1000–$1500, egg banking or embryo storage costs $300–$500 a year in storage fees, and IVF may cost $15,000–$20,000  [In terms of the importance of fertility preservation, previous research suggests that whilst lower numbers of transgender (and in some research non-binary) people are already parents than would be expected from general population data, the numbers of transgender and non-binary people who wish to become parents in the future is not negligible –7. TheseWhilst there are increasing options available for transgender or non-binary people to undertake fertility preservation, there is still only a limited amount of international research exploring the views of transgender people on this topic, and even less with non-binary people. De Sutter et al.’s  groundbrIn terms of decision making about fertility preservation, studies have found that this can be influenced by perceived psychological and physiological challenges associated with either ceasing hormone treatments in order to undergo fertility preservation , or delaIs genetic relatedness valued, does this relate to undertaking fertility preservation, and how is genetic relatedness accounted for?How does gender-affirming medical treatment relate to fertility?How prevalent is fertility preservation and who is most likely to undertake it?What influences decision making about fertility preservation, and does decision making occur in the context of professional advice or counselling?Whilst previous research has included comment on decision making, as summarised above, this has occurred in the context of research on intentions, experiences and outcomes for transgender  people who plan to undertake, or who have already undertaken, fertility preservation. The present study sought to focus specifically on decision making about fertility preservation amongst a convenience sample of transgender and non-binary people, and was guided by the following research questions:The mixed-methods study design was intended as a scoping study given the relative lack of previous research specifically on decision-making. Ethical approval for the research was granted by the Flinders University Social and Behavioural Research Ethics Committee (project number 7867).Participants were recruited via emails to Australian organisations and groups made up of and/or working with people who are transgender or non-binary, including broader ‘LGBT’ organisations. Organisations and groups were asked to share information about the questionnaire via their social media (Facebook and/or Twitter) and via an email to their members. Thirty-five organisations and groups were contacted and approximately half shared the details of the questionnaire. The researchers did not have access to member names or email addresses. The questionnaire was open from January–February 2018. No financial incentive was offered to participants.To be included in the final sample, at minimum participants needed to have answered socio-cultural demographic questions, and whether or not they had undertaken fertility preservation. In total 442 people commenced the questionnaire. Of those, 28 people started the questionnaire but did not respond to any items. An additional five people started the questionnaire but did not complete the minimum questions required. Of the final sample of 409 participants, participants on average spent 12 min completing the questionnaire.Participants were invited to complete an online questionnaire hosted on SurveyMonkey. No identifying information  was collected as part of the questionnaire, and the IP addresses of participants were not tracked. SurveyMonkey servers use secure encryption, and access to the data collected is available only to the researchers . The first page of the questionnaire included background information to the research and links to a full information sheet and a list of support resources. Participants were asked whether they consented to participate in the questionnaire by selecting ‘Yes’ or ‘No’ after reading the information on the first page of the questionnaire. Participants selecting ‘Yes’ were directed to the start of the questionnaire. Those selecting ‘No’ were redirected to a page welcoming them to return and complete the questionnaire at another time.The questionnaire, designed by the authors, drew upon previous research to focus on key variables which are likely to shape decision making around fertility preservation . The quParticipants who indicated they had undertaken fertility preservation were asked an open-ended question about how they had made this decision, whether or not they had received advice or counselling prior to their decision, whether they had delayed their gender transition in order to undertake fertility preservation, and how likely they were to use stored gametes in the future . Participants who indicated they had not undertaken fertility preservation were asked an open-ended question about how they made this decision, whether they had received any advice or counselling prior to making this decision, and whether they would have considered delaying their gender transition in order to undertake fertility preservation. Both of these pages concluded by asking whether participants thought that fertility preservation should be available to all transgender and non-binary people. In sum, participants who indicated they had undertaken fertility preservation were presented with a total of 15 questions to answer, and participants who indicated they had not undertaken fertility preservation were presented with 14 questions to answer.Questionnaire data were exported from SurveyMonkey into SPSS 21.0. Descriptive statistics were generated for socio-cultural demographics  having children already (or not) and gender, 2) views on the importance of genetic relatedness and fertility preservation, 3) age and gender-affirming treatments, 4) gender and having undertaken fertility preservation, 5) gender and willingness to delay gender transition in order to undertake fertility preservation, and 6) gender and the wish that fertility preservation had been undertaken. Additional tests were run on any relationship between advice or counselling received about fertility preservation and gender. Log-likelihood ratio tests (LLR) were used for all of the inferential statistics involving two categorical variables, given the existence of small cell sizes. For each LLR percentages are reported for each category so as to provide the reader with a breakdown of differences between groups. For the analysis of variance, Levene’s Test of Equality of Variance was used to test the assumption of equal variances, and to test the linearity of the data the Lack of Fit test was used. For each, results were non-significant, indicating that there were equal variances across groups examined, and that the data were linear.Open-ended responses were analysed via a conventional content analysis, following the guidelines outlined by Hsieh and Shannon . This inM = 28.54, SD = 11.25). Other socio-cultural demographics are outlined in Table Participants ranged in age from 18 to 72 years  = 53.97, P < 0.001. For participants who were not parents, 33% desired to have children in the future, of whom 44% were non-binary 36% were male, 12% were female, and 8% were agender, LLR  = 47.87, P < 0.001.In terms of having children, 64 of the participants were already parents, and 345 participants were not  = 15.574, P = 0.008.In terms of views on the importance of sharing a genetic relationship with children, 244 participants responded to this question, of whom 82 (33.6%) thought that genetic relatedness was important, and 162 (66.4%) thought it was not important. Focusing specifically on fertility preservation, of those who had undertaken fertility preservation 71% thought that genetic relatedness was important, and for those who had not undertaken fertility preservation 31% thought that genetic relatedness was important, not important to them. The most common response of these three categories was that participants were planning to adopt or foster, and thus had no interest in genetic relatedness. The other three categories pertained to participants who responded that genetic relatedness was important to them. The most common response of these three categories was a pragmatic acceptance that genetic relatedness was not possible, even if desired.With regard to the open-ended question about the importance of genetic relatedness, a content analysis of the 91 responses to this question identified six key categories, as outlined in Table F  = 14.49, P < 0.001. Post hoc comparisons using the Bonferroni test indicated that the mean age for participants who had undertaken gender affirming surgery was higher  than the mean ages for puberty blockers , current use of hormones , and past use of hormones . Also outlined in Table Participants were asked about any gender-affirming medical treatment they had undertaken which may impact on fertility. Responses are outlined in Table SD = 8.29). The mean for likelihood of using stored gametes was 2.28 (SD = 1.24), meaning that participants reported that they were only somewhat likely to use their stored gametes in the future. Of the 335 participants who responded to the question about whether or not fertility preservation should be offered to all transgender and non-binary people, 317 (94.6%) answered yes.Table LLR  = 13.910, P < 0.001. In terms of delaying gender transition in order to have undertaken fertility preservation, of those who answered yes 66% were female, 20% were male, and 14% were non-binary, LLR  = 14.806, P = 0.006. With regard to advice or counselling received prior to undertaking fertility preservation, there were no statistically significant differences in terms of gender. For those who had not undertaken fertility preservation, a majority (68%) indicated that they had not received any advice or counselling about fertility preservation. Of those who had not received advice or counselling, 39% were non-binary people, 30% were male, 20% were female, and 11% were agender, LLR  = 77.625, P = 0.005.Of those who had undertaken fertility preservation, 53% were female, 21% were non-binary, 18% were male, and 8% were agender, . As reported in Table . Of the participants who had not undertaken fertility preservation, 155 responded to an open-ended question asking about how they had made this decision. A content analysis of these responses indicated six categories, as outlined in Table In terms of decision making for the 28 participants who had undertaken fertility preservation, a content analysis of the 22 open ended responses indicated that participants primarily focused on who helped them make the decision, rather than why they made the decision per seDrawing on a convenience sample of Australian transgender and non-binary people, this mixed-methods study found that only a minority of participants viewed being genetically related to children as important, though participants who had undertaken fertility preservation were more likely to hold this view. The relative lack of importance placed upon genetic relatedness amongst the sample echoes previous research with transgender (and in some studies non-binary) people in regards to fertility preservation and parenting –10, thouOnly a small number of participants had undertaken fertility preservation, and a majority of these participants had delayed their gender transition in order to preserve their fertility. For the majority who had not undertaken fertility preservation, delaying gender transition in order to preserve fertility was not considered. The relatively small number of participants who had undertaken fertility preservation also echoes previous research –15, as iIn terms of receiving advice or counselling about fertility preservation, over half of those who decided to preserve their fertility had received advice or counselling. For those who had not undertaken fertility preservation, less than a quarter had received advice or counselling. Previous research has paid little attention to whether advice or counselling is offered in the context of fertility preservation, despite the recommendations of the WPATH SOC that health care professionals discuss reproductive options prior to medical treatment. This finding is thus a novel contribution of the research reported in this paper.The strength of the present study is that it represents the largest study to date of fertility preservation decision making undertaken with a non-clinical sample of transgender and non-binary people. The mixed-methods approach allows for an understanding of both the rates at which fertility preservation occurred amongst the sample and aspects of decision making related to this, as well as something of the meaning that participants attributed to decision making. The inclusion of non-binary people in the present study adds an important dimension to previous studies that have focused primarily on transgender people. In terms of limitations, the participants were all Australian and were comprised of a convenience sample, meaning that the findings may not be generalizable to other contexts. Additionally, responses to the open-ended questions around decision-making were on average relatively brief, meaning that more in-depth analyses of this qualitative data were not possible.Future research, including that planned as part of the present study, will benefit from focusing on experiences with fertility preservation. Whilst there are a small number of publications focused on this topic , 17, 22,not received advice or counselling about this were non-binary people. This suggests the need for greater awareness about the importance of discussing fertility preservation options with non-binary people, both amongst clinicians who work with non-binary people, and non-binary people themselves. Finally, and given that the average age of participants in the present study who had undertaken fertility preservation was relatively young, this suggests that whether or not encouraging fertility preservation is useful will likely depend on the age of the person.Given that the WPATH SOC  are currIn terms of the wider clinical relevance of the findings, it is suggested that a focus on the meaning or importance of genetic relatedness may be a useful heuristic through which to provide counselling to transgender and non-binary people considering fertility preservation. This point may be especially salient given that those who had undertaken fertility preservation reported that they were on average only somewhat likely to use stored gametes in the future. Considering the high costs of fertility preservation including ongoing storage fees , encouraging all transgender and non-binary people to undertake fertility preservation may not necessarily indicate best outcomes. Given the many pathways to parenthood available to transgender and non-binary people, other options besides genetic relatedness may be explored in order to mitigate the costs of fertility preservation if genetic relatedness is not viewed as important.


TSLP confers apoptosis resistance via concomitant activation of the TSLP/ signal transducer and activator of transcription (STAT)-5 / myeloid cell leukemia (Mcl)-1 route and a newly uncovered TSLP/ c-Jun-N-terminal kinase (JNK)/ B-cell lymphoma (Bcl)-xL axis, as evidenced by RNA interference and pharmacological inhibition. Our findings highlight the potential contribution of TSLP to the MC supportive niche of the skin and, vice versa, highlight MCs as crucial responders to TSLP in the context of TSLP-driven disorders.Mast cells (MCs) play critical roles in allergic and inflammatory reactions and contribute to multiple pathologies in the skin, in which they show increased numbers, which frequently correlates with severity. It remains ill-defined how MC accumulation is established by the cutaneous microenvironment, in part because research on human MCs rarely employs MCs matured in the tissue, and extrapolations from other MC subsets have limitations, considering the high level of MC heterogeneity. Thymic stromal lymphopoietin (TSLP)—released by epithelial cells, like keratinocytes, following disturbed homeostasis and inflammation—has attracted much attention, but its impact on skin MCs remains undefined, despite the vast expression of the TSLP receptor by these cells. Using several methods, each detecting a distinct component of the apoptotic process  Mast cells (MCs) are tissue-resident key effector cells of Immunoglobulin E (IgE)-mediated allergic and inflammatory responses, including common skin disorders, such as atopic dermatitis (AD) and psoriasis ,3,4,5,6.Thymic stromal lymphopoietin (TSLP) is mostly expressed by epithelial cells, including keratinocytes ,19,20. TWhile the responsiveness of MCs to TSLP has been occasionally reported ,26,27,28L upregulation. These two well-known antiapoptotic Bcl-2 proteins jointly organize the survival of skin MCs.Here, we identify TSLP as a pro-survival factor of human skin MCs. We kinetically resolve the sequence of events underlying TSLP’s antiapoptotic action. Initially, TSLP activates the signal transducer and activator of transcription (STAT)-5 and c-Jun-N-terminal kinase (JNK), which are both implicated in survival prolongation. STAT5 acts by enhancing myeloid cell leukemia (Mcl)-1, while JNK is responsible for B-cell lymphoma (Bcl)-xMCs were isolated from human foreskins, whereby material from several donors  was combined for one experiment to achieve the cell numbers required, as described ,32,33. TSkin MCs were used either ex vivo or cultured in Basal Iscove medium, supplemented with 10% fetal calf serum , SCF  (at 100 ng/mL), and interleukin (IL)-4 (Peprotech) (10 ng/mL) ,38,39.Skin fibroblasts and keratinocytes, used as controls, were isolated according to routine protocols ,41Skin MCs were treated with different concentrations of TSLP (Peprotech) for the indicated times in minimal medium, consisting of Basal Iscove medium , supplemented with 0.5% bovine serum albumin (BSA) . After incubation, cells were processed for downstream applications (as described below).5 cells/mL) were pre-incubated for 15 min, with pimozide , SCH772984 1/2 inhibitor, 5 µM) , SB203580 , or SP600125  , prior to the addition of TSLP. After incubation, cells were processed for downstream applications (see reverse transcription - quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and analysis of survival).For kinase inhibition studies, cells (at 5 × 105 cells/mL) were treated with different concentrations of TSLP in minimal medium for the indicated times and processed for survival detection by different methods, based on our previously established protocols [MCs  and propidium iodide  for 30 min on ice. YoProTM-1 staining detects membrane porosity and, combined with PI, can serve to distinguish apoptotic cells from viable and necrotic cells.For YoProApoptosis detection by annexin V-fluorescein isothiocyanate (FITC)/PI (BD Biosciences) was performed according to the protocol of the supplier. In brief, cells were stained with annexin V-FITC and PI in annexin V binding buffer (BD Biosciences) for 20 min at room temperature (RT). Annexin V-FITC detects the externalization of phosphatidylserine and, combined with PI, can distinguish apoptotic from viable and necrotic cells.TM-1/PI and annexin V-FITC//PI, double-positive cells were considered cells in advanced apoptosis or necrosis, whereas YoProTM-1- or annexin V-FITC-positive but PI-negative cells were considered early apoptotic. Double-negativity was indicative of viable cells. YoProTM-1 or annexin V-FITC positivity was calculated as the sum of the percentages displayed in quadrant (q)1 and q2, based on the values given in For YoProFor PI staining, cells were treated with PI  for 1 h at 4 °C, which was performed to detect the percentage of hypodiploid nuclei corresponding to cells with fragmented DNA. The cells were measured on a MACS Quant FACS (fluorescence-activated cell sorter)  and analyzed using the FlowJo software .® 3/7; Promega, Mannheim, Germany), providing a proluminescent caspase-3/7 substrate, which is cleaved to release luminescence, which was detected by means of a microplate reader , as described [The caspase-3 activity of MCs was measured using a luminometric assay kit (Caspase-Gloescribed .TM-1 and caspase-3) could be unambiguously detected (not depicted). This served to ensure a predominance of direct effects and limit secondary effects . We found this period to be 8 h.Prior to perturbation experiments, kinetics series were performed to identify the shortest incubation period, after which survival promotion by TSLP (by YoProTM-1 positivity (1) or caspase-3 activity (2) in TSLP-treated versus untreated MCs was calculated by the following formulas in perturbation experiments. Based on the values given in Figures 3 and 5a, with early-apoptotic, late-apoptotic/necrotic, PI-single positive necrotic, and viable cells being found in q1, q2, q3, and q4, respectively, the calculation was as follows:Using this period, the ratio of YoPro% Rescue effect of TSLP =5 cells/mL) were deprived of GF (growth factors)/serum for 16 h in minimal medium. After starvation, cells were incubated for a further 15 min with TSLP, then fixed with 4% paraformaldehyde (PFA) for 30 min at RT and permeabilized with 0.1% Saponin for 20 min at 4 °C. Cells were stained with anti-pSTAT5 or anti-pJNK primary antibodies  (or the respective isotype control) for 30 min on ice, followed by incubation with Phycoerythrin (PE)-labeled secondary antibodies  for 30 min on ice.For the intracellular staining of signaling intermediates, skin MCs  and analyzed using the FlowJo software (FlowJo LLC). Data were shown as net mean fluorescence intensity (MFI), as calculated by the following equation:® siRNA transfection technology . MCs were transfected by gene-targeting siRNA or non-targeting siRNA (serving as control). Briefly, MCs were washed with 1X Accell siRNA medium , plated at 106/mL in Accell siRNA medium, and treated with 1 µM STAT5-targeting siRNA (E-005169-00-0010), JNK-targeting siRNA (E-003514-00-0010), Mcl-1-targeting siRNA (E-004501-00-0010), Bcl-xL-targeting siRNA (E-003458-00-0010), or non-targeting siRNA (D-001910-10-50) for 48 h. After incubation, cells were treated with TSLP for the indicated times and processed for downstream applications .RNA interference in MCs was performed according to a recently established protocol , using t5 cells/mL) were deprived of GF/serum for 16 h in minimal medium. After starvation, cells were incubated with TSLP for the indicated time points. RT-qPCR was performed as described, using primers described therein [® Green kit .Skin MCs  for SCF and IL-33 were as follows:The expression levels of the target gene were quantified relative to the expression of the reference gene SCFForwardGCGTGGACTATCTGCCGCCGReverseAGCGCTGCGATCCAGCACAAAIL-33ForwardTGTCAACAGCAGTCTACTGTGGReverseTGGACCCCTGATATACCAAAGG5 cells/mL) were deprived of GF/serum for 16 h in minimal medium. To study the phosphorylation of signaling molecules, cells were incubated for a further 30 min with TSLP. As positive controls, we used a combination of SCF (10 ng/mL) and IL-33 (20 ng/mL) for pERK and pp38, and human mast cell line (HMC)-1 cells (3 × 105 cells per lane), kindly provided by Dr. J.H. Butterfield [L protein expression, cells were incubated with (or without) TSLP for 2 h or 4 h. After incubation, MCs were lysed and separated through 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [L, phospho/total-ERK, phospho/total-p38, phospho/total-JNK, phospho/total-STAT3, and phospho/total-STAT5, as well as ß-actin and Cyclophilin B as loading controls (each diluted 1:1000) , overnight, and subsequently with  HRP (horseradish peroxidase)-conjugated secondary antibodies  for 1.5 h, as described [Skin MCs  ,39. The escribed ,31,32. Ft-test. For normalized data, the Wilcoxon matched-pairs signed rank test was used . In P-values less than 0.05 were considered statistically significant. Data were analyzed with GraphPad Prism Version 6.01 Software .Results are reported as mean ± standard error of the mean (SEM). In TM-1 dye permeates the slightly porous membrane of apoptotic cells and thus enables the visualization of phosphatidylserine (P-ser)-independent membrane alterations indicative of apoptosis. Using this method, TSLP-treated MCs displayed 44% less YoProTM-1 positivity  and prositivity b.Addressing DNA degradation, a characteristic feature of apoptosis in its later stage , we founTogether, TSLP protects human skin MCs from apoptosis, as evidenced by different readouts.Next, we addressed the downstream events triggered by TSLP in skin MCs. This was performed by studying the phosphorylation of STAT3, STAT5, and the three MAPKs JNK, ERK1/2, and p38. Both JNK and STAT5 were phosphorylated on the respective activatory sites following TSLP treatment a,e. ThisTM-1 positivity  of the respective component by RNA interference (RNAi). The latter approach revealed that survival promotion by TSLP indeed depends on STAT5, as the antiapoptotic effect decreased from 35% (control) to 10% after STAT5 KD, as assessed by YoPrositivity a. The sasitivity a. Targetsitivity b. For KDsitivity , whereasout TSLP .Specific inhibitors supported the above findings. Pimozide (STAT5 inhibitor) led to a decrease from 34% to 4% c, while Together, interference with STAT5 and JNK impeded TSLP from exerting its anti-apoptotic effect, implying key roles for these components in the antiapoptotic machinery contracted by TSLP.Mcl-1 and LBcl-x mRNA expression at both 40 and 90 min  to 8% (Mcl-1-silencing) or 9% (Bcl-xL-silencing)  a. In thelencing) , while tsurvival . Taken tL, are required for survival prolongation by TSLP. We were finally interested in unraveling the relationships between the early and the later events. To this end, the TSLP modulation of Mcl-1 and Bcl-xL levels was monitored upon interference with STAT5 and JNK function. While STAT5 perturbation completely blocked Mcl-1 upregulation by TSLP  [It was important to assess whether TSLP protects against MC apoptosis directly upon their isolation from skin tissue. In fact, several differences exist between skin MCs ex vivo and upon long-term pre-culture ,44,48. TCRLF2) .TM-1-positive cells  yielded congruent results, i.e., caspase-3 activity (very low in ex vivo MCs) was attenuated b, and sove cells c and subve cells d. ConsidChronic inflammation is typically associated with increased numbers of MCs in the respective tissues, where the cells are believed to contribute to the underlying disease ,2,4,50. L upregulation, whose joint action finally impedes skin MC demise. Our results highlight the parallel existence of a STAT5–Mcl-1 and a JNK–Bcl-xL axis in human skin MCs.Here, we identified a new survival promoter of human skin MCs and revealed the mechanisms of its action. TSLP operates by the initial activation of STAT5 and JNK, which subsequently leads to Mcl-1 and Bcl-xL were upregulated already at 40 min  to mt 40 min . Second,t 40 min , and thi and low . The antTC subset, whereas most MCs in the lung are of the MCT type [TC and MCT MCs in their response pattern to TSLP. The TSLP/TSLPR axis has been indicated to strengthen the MC compartment also in vivo in humans and mice [TSLP notably contributes to skin disorders like AD ,54, and MCT type ,30, the and mice ,55.Our findings with freshly isolated dermal MCs  strengthAddressing the mechanisms, we first found that STAT5 and JNK are both phosphorylated and implicated in TSLP-conferred apoptosis resistance, as their ablation or inhibition impaired survival promotion.Although TSLP can trigger disparate signal transduction pathways, depending on the cell type , the actOur finding that STAT5 contributes to TSLP’s survival machinery in skin MCs is consistent with a long series of studies, demonstrating its relevance in the MC lineage ,68,69,70In contrast, JNK is described as a “double-edged sword”, either promoting or inhibiting apoptosis, depending on the precise conditions ,72. In sL, Mcl-1, and proapoptotic entities, including Bad, Bax, Bak, and Bid, crucially controls the mitochondrial pathway of apoptosis. Their participation in the regulation of MC survival has been described in multiple reports [We then addressed the potential downstream targets of STAT5 and JNK. The Bcl-2 family, comprising antiapoptotic members like Bcl-2, Bcl-x reports ,74,75,76L and Mcl-1 were distinctively upregulated by TSLP. To clarify their individual roles in survival promotion, we employed RNA interference to deplete Mcl-1 and Bcl-xL expression prior to stimulation with TSLP. Interestingly, Mcl-1 and Bcl-xL both equally imparted TSLP protection. This is in line with previous reports demonstrating that Mcl-1 is tightly connected to death/survival decisions of the lineage in transgenic mice [We found that Bcl-xnic mice , and in nic mice  and humanic mice ,76,79. Inic mice , a functnic mice . Upon cuL as another factor controlling MC survival in the presence of TSLP. This antiapoptotic molecule has likewise been widely documented as a survival promoter of MCs, however primarily in response to IL-33, SCF, and IgE receptor activation [KD experiments identified Bcl-xtivation ,74,75,76L, we sought to examine potential links between the proximal events (STAT5 and JNK activation) and the subsequent upregulation of these factors. Intriguingly, we found a clear-cut distinction between the Bcl-2 family members, as inhibition of STAT5 counteracted Mcl-1 with no impact on Bcl-xL expression, whereas JNK perturbation resulted in decreased Bcl-xL upregulation without affecting Mcl-1 expression, speaking in favor of well separated pathways. In fact, Mcl-1 can be activated by STAT5 and regulate survival in different cells [L has also been reported as a target of STAT5 in other cell types [L was even found to be under negative influence from JNK in a number of other cells [After revealing the crucial roles of Mcl-1 and Bcl-xnt cells ,81,82. Bll types ,84, the er cells ,86.We assume that the simultaneous upregulation of both factors is necessary to exceed a certain “antiapoptotic” threshold to allow for TSLP’s protective function to come into force. In this scenario, TSLP would only counteract cell death in the event of both antiapoptotic Bcl-2 members jointly reaching this point. This theory is supported by findings showing that mitochondrial membrane disruption and concomitant cytochrome c release can depend on a distinct threshold of mitochondrial membrane permeabilization .L, and consequent apoptosis resistance. The increased lifespan is thus owed to the concomitant activation of two axes, namely STAT5/Mcl-1 and JNK/Bcl-xL. Our findings support a role for TSLP as a crucial component of the MC supportive niche in lesional skin, and its overabundance during disturbed homeostasis may help explain MC accumulation in cutaneous pathology.Taken together, we uncovered TSLP as a novel survival factor of human skin MCs, which exerts its antiapoptotic effect by the synchronized activation of STAT5 and JNK, the ensuing segregated upregulation of Mcl-1 and Bcl-x


US Jersey cattle. The dataset included 1.5 K Jersey bulls with sire conception rate (SCR) records and 96 K single nucleotide polymorphism (SNP) markers spanning the whole genome. The analysis included whole‐genome scans for both additive and non‐additive effects and subsequent functional enrichment analyses using KEGG Pathway, Gene Ontology (GO) and Medical Subject Headings (MeSH) databases. Ten genomic regions located on eight different chromosomes explained more than 0.5% of the additive genetic variance for SCR. These regions harbor genes, such as PKDREJ,EPB41L2,PDGFD,STX2,SLC25A20 and IP6K1, that are directly implicated in testis development and spermatogenesis, sperm motility and the acrosome reaction. In addition, the genomic scan for non‐additive effects identified two regions on BTA11 and BTA25 with marked recessive effects. These regions harbor three genes—FER1L5,CNNM4 and DNAH3—with known roles in sperm biology. Moreover, the gene‐set analysis revealed terms associated with calcium regulation and signaling, membrane fusion, sperm cell energy metabolism, GTPase activity and MAPK signaling. These gene sets are directly implicated in sperm physiology and male fertility. Overall, this integrative genomic study unravels genetic variants and pathways affecting Jersey bull fertility. These findings may contribute to the development of novel genomic strategies for improving sire fertility in Jersey cattle.The service sire has been recognized as an important factor affecting herd fertility in dairy cattle. Recent studies suggest that genetic factors explain part of the difference in fertility among Holstein sires. The main objective of this study was to dissect the genetic architecture of sire fertility in  Reproductive management of dairy cows has advanced significantly in the past 20 years with the advent of ovulation synchronization protocols, improvements in nutrition and cow comfort and, more recently, the incorporation of fertility and longevity traits into genetic selection programs. However, despite these advances, reproductive efficiency of dairy cattle remains suboptimal, resulting in significant economic losses for the dairy industry . This bull fertility evaluation is based on cow field data, considering both factors related to the sire under evaluation (e.g. age and AI stud) and factors associated with the cow that receives the unit of semen  .The entire evaluation of US Jersey bull fertility was used in this study. Specifically, a total of 6353 SCR records were available from 1557 Jersey bulls. These records were obtained from 27 consecutive SCR evaluations between August 2008 and August 2017. The SCR records and their reliabilities, calculated as function of the number of breedings, are freely available at the CDCB website (Genotype data for 107 371 single nucleotide polymorphism (SNP) markers were available for 1487 Jersey bulls with SCR evaluation. Genotype data were kindly provided by the Cooperative Dairy DNA Repository. The SNP markers that mapped to the sex chromosomes, were monomorphic or had minor allelic frequency less than 1% or calling rate less than 90% were removed from the dataset. After quality control, a total of 95 705 SNP markers were retained for subsequent genomic analysis.et al. et al. y is the vector of SCR records, β is the vector of fixed effects included in the model, u is the vector of the random animal effects, pe is the vector of random permanent environmental and non‐additive effects and e is the vector of random residual effects. The matrices X, Z and W are the incidence matrices relating phenotypic records to fixed, animal and permanent environment effects respectively. The random effects are assumed to follow a multivariate normal distribution, σu2, σpe2 and σe2 are the animal additive genetic, permanent environmental and residual variances respectively. The classical pedigree relationship matrix A is replaced by H, which combines pedigree and genotypic information  was calculated based on a five‐generation pedigree downloaded from the CDCB website. The residual matrix R is a diagonal matrix with its elements representing the reliabilities of the SCR values.The relevance of additive effects was investigated using the single‐step genomic best linear unbiased prediction (ssGBLUP) method (Aguilar û), the SNP effects can be estimated as s^=DM′[MDM′]−1u^, where M is a matrix relating genotypes of each locus and D is a diagonal matrix of weights of SNPs  and e∼N. The matrices G2 and I have dimensions 1487 × 1487, i.e. the total number of Jersey bulls with both phenotypic and genotypic data. The variance–covariance matrix of this model was estimated as V0=ZG2Z′σu2+Iσe2.In the first step, the following mixed model was fitted: y = Xβ + XSNPβSNP + ϵ, assuming e∼N. Every SNP genotype XSNP  was recoded using single numeric variables as ,  and  for testing potential dominance, recessive and overdominance effects respectively. The significance of each non‐additive effect was tested using the following test statistic: mixabel   jointly explained 1.4% of the additive genetic variance. Remarkably, these two regions harbor several candidate genes for bull fertility, including ZMYND10 and SLC25A20, which are directly involved in sperm motility; IP6K1 and RBM5, which play critical roles in male germ cell maintenance and differentiation; and PDHB, which is implicated in sperm capacitation. Moreover, two 1.5‐Mb SNP windows located on BTA1 (14.1–15.6 and 90.1–91.6 Mb) explained a substantial amount of the genetic variance (1.3%); these regions harbor genes NCAM2 and TBL1XR1, which are involved in testis development and spermatogenesis. In addition, three regions distributed on BTA5 (117.6–119.1 Mb), BTA9 (69.9–71.4 Mb) and BTA15 (3.7–5.2 Mb) explained roughly 0.65% of the additive genetic variance. At least three genes directly implicated in sperm physiology and male fertility, namely PKDREJ, EPB41L2 and PDGFD, are located in these three regions respectively. Finally, the region identified on BTA17 (45.9–47.4 Mb) harbors two candidate genes: DDX51, which is involved in spermatogenesis and testis development, and STX2, which plays an active role in the acrosome reaction.The importance of additive effects on SCR was evaluated using ssGBLUP. This method was originally developed for genomic prediction and later was extended for performing gene mapping. The proportion of the additive genetic variance explained by 1.5‐Mb SNP windows across the entire bovine genome is shown in Fig. P‐value < 0.01) , PDGFD  and IP6K1 , are directly implicated in testis development and spermatogenesis. EPB41L2 encodes a membrane protein expressed in Sertoli cells that mediates cell–cell contact between Sertoli cells and germ cells during spermatogenesis  and STX2 —are directly involved in the acrosome reaction, a crucial step during the fertilization. PKDREJ encodes a sperm surface receptor that modulates G protein signaling and mediates sperm–egg interaction (Hamm et al. STX2 encodes a member of the SNARE protein family that controls the membrane fusion during the sperm acrosome reaction (Hutt et al. ZMYND10 and SLC25A20—located on BTA22 at 50.3–51.8 Mb, are directly implicated in sperm motility. ZMYND10 is almost exclusively expressed in the testis, plays a key role in cilia integrity and has been implicated in sperm dysmotility and male infertility (Moore et al. SLC25A20 encodes a carnitine carrier in the mitochondrial membrane that is involved in ATP production and cell energy metabolism and has been associated with human asthenozoospermia, aka low sperm motility (Asghari et al. The ssGBLUP method revealed 10 genomic regions on seven different chromosomes that explain significant amounts of additive genetic variance. Interestingly, most of these regions harbor genes with functions associated with male fertility. For instance, three putative genes related to Jersey sire fertility, namely et al. FER1L5, CNNM4 and DNAH3, which have direct roles in male fertility. FER1L5 encodes a member of the ferlin family that regulates calcium‐mediated membrane fusion events during spermatogenesis (Washington & Ward CNNM4 encodes a magnesium transporter that regulates calcium homeostasis during the sperm capacitation and is essential for ensuring sperm fertilizing ability (Yamazaki et al. DNAH1 gene, which encodes a member of the dynein family, cause multiple abnormalities of the sperm flagella, resulting in impaired sperm motility and, hence, male infertility (Ben Khelifa et al. It is believed that non‐additive effects are relevant for fitness‐related traits such as reproduction. Indeed, our recent study revealed significant dominance effects for SCR on Holsteins (Nicolini et al. et al. et al. et al. et al. (Genomic scans typically detect only major regions, while the vast majority of the genetic variants remain hidden. Therefore, complementary approaches are needed to fully reveal the genetic architecture underlying a complex phenotype. Here, alternative gene‐set analyses were performed to identify biological processes and molecular mechanisms responsible for the SCR variation in US Jersey bulls. Interestingly, gene sets directly related to calcium regulation and calcium signaling were among the most significant functional terms. Calcium is implicated in different sperm physiological responses including sperm maturation, sperm motility and sperm capacitation (Darszon . et al. , these set al. It should be noted that none of the ten 1.5‐Mb SNP windows nor the two recessive SNP loci were previously reported as significantly associated with bull fertility in Holstein cattle. This may be due to multiple causes: the major mutations affecting bull fertility in Jersey are not segregating in Holstein, these causative mutations are indeed segregrating in Holstein but are not in high linkage disequilibrium with the SNP markers or simply false‐positive/false‐negative results. However, most of the gene sets significantly associated with SCR in Jersey were also identified as relevant functional terms affecting Holstein bull fertility (Peñagaricano We performed an integrative genomic analysis to dissect the genetic basis of SCR in US Jersey dairy cattle. Ten different regions distributed on BTA1, BTA5, BTA9, BTA11, BTA13, BTA15, BTA17 and BTA22 explained significant amounts of additive genetic variance. In addition, two regions on BTA11 and BTA25 showed marked recessive effects. Interestingly, most of these genomic regions harbor genes that play key roles in male reproduction, including testis development, spermatogenesis, sperm motility and fertilization process. Moreover, the gene‐set analysis revealed functional gene sets, such as calcium signaling, calcium channels, membrane fusion, pyrophosphatase activity, GTPase activity and MAPK signaling, that are directly related to spermatogenesis, sperm cell energy metabolism and acrosome reaction. Overall, this comprehensive study unraveled genetic variants and biological pathways responsible for the variation in Jersey bull fertility. Our findings can provide opportunities for improving service sire fertility in Jersey cattle via marker‐assisted selection.


Dear Editor,Although the interplay between tumor progression and blood coagulation has been recognized since the milestone works by Bouillaud and Trousseau ,2, the mIn the first part of the paper, EPCR and PAR-1 canonical signaling pathways are detailed . EndotheMore interestingly, the authors give insights into other “hidden” functions of EPCR that place this receptor at the interface between hemostasis and inflammation. Besides endothelial cells, EPCR is expressed on a variety of cell types, including dendritic cells , leukocy2+ signaling by removing the N-terminal tethered ligand from the PAR-1 receptor [Along with EPCR, in recent years, novel functions have been emerging for PC/APC, placing it center stage in cellular signaling. Once in its active form, APC may exploit the canonical anti-coagulant and fibrinolytic functions or—in complex with EPCR—it may attack and activate the PAR-1 receptor. The unexpected role of the APC–EPCR complex in PAR-1 proteolysis was uncovered by Wolfram Ruf and co-workers , and sinreceptor . Besidesreceptor . Interesreceptor . This opreceptor . Variousreceptor .While the expression profile of PARs in different tissues is well defined, the tissue-specific signaling function of these receptors is poorly resolved. In particular, while there is a wealth of knowledge on PAR-1 signaling in endothelial cells and its regulatory role in vascular permeability and tumor intravasation , it is i14 microorganisms colonizing the gastrointestinal tract is a non-negligible environmental factor, modulating multiple signaling pathways. Besides its trophic and metabolic functions, the gut microbiota has an impact on cancer initiation, progression, and even on anticancer therapies [Helicobacter pylori infection is a significant risk factor of stomach cancer) [A fascinating aspect that needs further investigation is the effect of the commensal gut microbiome on tumor growth and dissemination. The huge diversity of the 10herapies ,43,44. R cancer) ,46. In t cancer) . Vice ve cancer) .Enterococcus faecalis gelatinase [Gut resident bacteria could potentially affect the APC/EPCR/PAR-1 axis by modulating PARs (i) expression and (ii) activation. In the small intestine, the gut microbiota is known to upregulate PAR-1 expression and to trigger tissue factor-dependent coagulation factor signaling . Proteaslatinase , or indilatinase ,51.In conclusion, the review article by Wojtukiewicz and co-workers  describe


Cancer treatments may compromise the fertility of children, adolescents, and young adults, and treatment-related infertility represents an important survivorship issue that should be addressed at diagnosis and in follow-up to ensure optimal decision-making, including consideration of pursuing fertility preservation. Risk of infertility varies substantially with patient and treatment factors. The ability to accurately assess fertility risk for many patients is hampered by limitations of the current literature, including heterogeneity in patient populations, treatments, and outcome measures. In this article, we review and synthesize the available data to estimate fertility risks from modern cancer treatments for both children and adult cancer survivors to enable clinicians to counsel patients about future fertility. Over the past decades, therapeutic advances have transformed the care of cancer patients, yielding substantial improvements in cure rates and survival. Children and young adults have gained particular benefit, given successes realized in the treatment of childhood acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (HL), and testicular cancer, among other malignancies. These gains have come with many costs, and, of the long-term treatment sequelae, infertility is among the most important to patients. The most comprehensive definition of “cancer survivor” includes patients from diagnosis onwards and recognizes that management of survivorship issues, even those arising years after treatment, must start at diagnosis . FertiliSeveral recent reviews of fertility preservation strategies have been published  and guidWe performed a review of published articles describing the risks of cancer treatments to female and male fertility using the PubMed database. Search terms included, but were not limited to, names of malignancies , cancer therapies , and outcomes . We included only peer-reviewed articles written in English. No date cutoff was imposed. Reference lists were reviewed for additional relevant articles. All study designs were considered. Randomized trials and prospective observational studies were included preferentially over retrospective studies. For the purposes of this review, the primary outcome of interest is fertility (the ability to conceive a child), and we thus report pregnancy and live birth data whenever possible and use surrogates, such as amenorrhea, semen parameters, and laboratory markers of gonadal insufficiency, as alternatives when necessary . FindingData from prior prospective and retrospective trials inform the risks of infertility, yet limitations exist in assessing the likelihood that an individual patient will remain fertile. Ovarian reserve varies among women and is affected by additional factors, including genetic polymorphisms associated with age of menopause  and otheDue to ease of measurement, amenorrhea is frequently reported as a primary outcome yet is an imperfect predictor of fertility. Female survivors who have spontaneous menses, particularly if irregular, may still have decreased ovarian reserve and reproductive potential . AbnormaThe following section details data regarding fertility risks in select populations, diseases, and treatments and is presented with a summary in >11.3 gm/m2, hazard ratio [HR] = 0.85, 95% CI = 0.74 to 0.98) , suggesting that most survivors treated with a variety of chemotherapy regimens alone are not at high risk of infertility (Multiple reports from the Childhood Cancer Survivor Study (CCSS), a multi-institutional long-term follow-up study that included patients and matched sibling control subjects, have demonstrated associations between chemotherapy use, as measured by summed alkylating agent dose or cyclophosphamide equivalent dose (CED), and risk of clinical infertility, never achieving pregnancy, and premature menopause . A 2016 ertility  30). Mo. Mo>11.3ertility , 31–33, ertility , 35. Theertility .Patient and disease factors appear to have little impact upon subsequent fertility outcomes among children. Age at diagnosis is an important predictor of fertility-related outcomes in some adult malignancies; however, data from the CCSS showed no association between age at diagnosis and future fertility . AlthougAmong adults, the most robust data regarding fertility risks are available for women with breast cancer. In a recent meta-analysis of five prospective, randomized trials evaluating gonadotropin-release hormone (GnRH) agonists during breast cancer chemotherapy, the rate of premature ovarian insufficiency in the control  arms was 31%, demonstrating that many premenopausal women retain ovarian function . ImportaP < .001)  or concurrently (TAC), in which 70% of participants were older than 40 years old, rates of TRA were lower with doxorubicin/docetaxel (38%) relative to AC-T (70%) and TAC (58%) ( < .001) . How mucAdditional data inform the risks of TRA with taxanes, which are now frequently included in adjuvant regimens. AC-T has been associated with numerically higher rates of TRA than AC alone , though neither difference was statistically significant , 47. In Neither dose-dense schedule nor the addition of trastuzumab has been associated with TRA , 55. PlaTamoxifen use following adjuvant chemotherapy is associated with a 2-fold increase in risk of TRA , 47, 60,TRA has been associated with improved overall survival in breast cancer ; thus, rIn the treatment of cervical cancer, the focus for women interested in future fertility has been on prevention of anatomical changes that impair childbearing. The risk of infertility with hysterectomy is 100%, although successful pregnancies have occurred with oocyte retrieval and use of a surrogate . FertiliAlthough hysterectomy with bilateral salpingo-oophorectomy represents the standard approach to localized endometrial cancer, continuous progestin therapy is a fertility-sparing option for highly selected young women with endometrial hyperplasia or stage IA endometrial adenocarcinoma . AlthougManagement of epithelial ovarian cancer also includes removal of critical reproductive organs. For highly selected women, depending on extent and type of disease, fertility-sparing procedures with unilateral salpingo-oophorectomy and complete surgical staging may be performed . In one Gonadal-sparing surgery is the goal of management of ovarian cyst and tumors in adolescent and young adult women. Most ovarian tumors in girls and adolescents are benign. Malignant tumors are usually of germ cell origin. For tumor marker negative tumors, a fertility-sparing procedure can be performed. With positive tumor markers, a unilateral oophorectomy and staging procedure—in which peritoneal fluid is collected, lymph nodes and omentum are inspected, and biopsy reserved for suspicious sites—is performed to maintain fertility. Minimizing abdominopelvic surgery prevents fertility issues due to adhesions. Most ovarian germ cell tumors in adolescents and young adults are stage I and about 50% are cured with surgery. For women requiring further treatment, fertility preservation should be considered, given the effect of cisplatin on ovarian function remains unknown in this setting.131I) may also be used for differentiated thyroid cancer and leads to transient amenorrhea in up to 20% of patients (131I (49.5 vs 51.0 years), but there is no difference in birth rate and 131I does not appear to have long-term effects on fertility  is nearly twice that of adjuvant breast cancer regimens, and therefore the effects on amenorrhea and fertility are expected to be at least that of women of corresponding age treated for breast cancer. However, young women with breast cancer have low rates of amenorrhea, and other studies suggest more favorable fertility outcomes for NHL survivors with greater than 50% achieving pregnancy and TRA in under 10%, even with dose-intensified CHOP (cyclophosphamide 12 000 mg/m2) (Non-Hodgkin lymphoma (NHL) encompasses indolent and aggressive diseases addressed with a variety of treatments, including chemotherapy, targeted therapies, and immune-based therapies. Some studies suggest relatively low fertility rates for female survivors, with successful pregnancies in 21%  and TRA 0 mg/m2) , 103. Ol0 mg/m2) . Women t0 mg/m2) . No dataThe backbone treatment for acute myeloid leukemia (other than acute promyelocytic leukemia) remains daunorubicin, an anthracycline, and cytarabine. Fertility risks associated with anthracyclines are poorly defined because anthracyclines have usually been co-administered with alkylators for more prevalent cancers. However, rates of amenorrhea with anthracyclines may be substantial even in the absence of alkylators . TreatmeThe risk to fertility associated with long-term imatinib, a tyrosine kinase inhibitor (TKI) targeting bcr-abl, for chronic myeloid leukemia is not well studied. Offspring of women exposed to imatinib during pregnancy have demonstrated congenital abnormalities, and patients should practice reliable contraception while on TKI therapy, including imatinib or others . Despiteper year 1.2, P = .002) and receipt of TBI  were associated with ovarian failure  relative to sibling control subjects . RadiatiPelvic radiation may also affect the uterus, leading to atrophy of the myometrium and endometrium  and decrCranial irradiation affects fertility through the development of endocrinopathies involving the hypothalamic-pituitary-gonadal axis . Among cInfertility among male cancer survivors is common yet has been the subject of less research than female infertility. Pregnancy and live birth data are sparse, and spermatogenesis parameters are frequently used as surrogates although generally felt to be more reliable than markers of ovarian reserve. Semen analysis methodology and reference ranges for semen volume, sperm concentration, total sperm number, morphology, and motility have been standardized , 139. EaAlthough treatment usually represents the greatest threat to fertility of male survivors, the underlying malignancy may affect semen production and quality through poorly understood mechanisms. In testicular cancer, rates of oligospermia and azoospermia at presentation are 50% and 10%, respectively . SimilarRadiation and chemotherapy have cytotoxic effects on testicular germinal epithelium including Sertoli cells, but to a lesser extent on Leydig cells, leading to frequent impairment of spermatogenesis without hypogonadism , 156. SpThe next section details data regarding the risk to male infertility in select populations, diseases, and associated treatment modalities and is presented with a summary in 2, HR = 0.89, 95% CI = 0.77 to 1.03; >7.4 gm/m2, HR = 0.60, 95% CI = 0.51 to 0.71), ifosfamide , and procarbazine , was associated with statistically significant decreases in male fecundity. Similar findings were demonstrated when analyzing by CED per 5-gm/m2 increment . Although lacking statistical significance, potentially due to inadequate statistical power, low or moderate exposure to the other alkylators, including busulfan  and lomustine , may still have clinically significant effects on male fertility. This builds upon prior evidence for a dose-dependent increase in risk of infertility and impairment in spermatogenesis among male cancer survivors treated with alkylating chemotherapy    than females  . Among motherapy . Among ato 1.34) . Importato 0.82) . Althougto 0.82) , 166.Although unilateral orchiectomy theoretically spares the unaffected testis, sperm concentration decreases post-procedure and azoospermia may occur among men who initially presented with oligospermia . TreatmeDespite data suggesting that testicular cancer survivors are as likely to sire a pregnancy as age-matched control subjects , adjuvan131I is associated with a testicular radiation exposure of about 0.10 Gy. No data regarding effects on spermatogenesis or fertility are available .Most male HL survivors will have normal fertility following treatment. Within a case-control study, 29.3% of survivors had biological children post-treatment relative to 32.4% of age-matched control subjects  . Among 3Over the past several years, therapy has shifted away from regimens heavy in alkylator exposure and towards ABVD. A prospective study that included HL and NHL found that no patients treated with ABVD were azoospermic 1 year post-treatment, with higher rates following CHOP (22%) and BEACOPP (75%) . Two yeaLimited data are available on risks to fertility among men receiving chemotherapy for acute leukemias. Patients with AML and ALL have been grouped together, despite differences in treatments, including in use of alkylating agents. One series of 13 patients with acute leukemias reported azoospermia in 46% , and twoPregnancies have been identified in female partners of males on imatinib therapy for chronic myeloid leukemia, though data are insufficient to conclude whether imatinib is associated with congenital abnormalities in offspring of males . The risConditioning chemotherapy and radiation frequently lead to severe impairment of male fertility. Fewer than 30% of men recover spermatogenesis and normal testicular function following HCT, with the lower rates of recovery with greater gonadotoxic exposures , 188–192The testes are sensitive to small radiation exposures, and the effect on spermatogenesis depends upon gonadal dose and radiation schedule . OligospGonadal exposure of 10 Gy in TBI yields azoospermia in more than 80% of patients, though concurrent alkylating chemotherapy may also contribute . Male chHigh-dose PCI (>24 Gy) is associated with diminished fertility among adult male survivors of childhood ALL. One analysis demonstrated impaired fertility for survivors treated with 24 Gy at ages younger than 10 years  and 10 years or older  . Within Young cancer survivors face wide variation in fertility risk attributable to age at diagnosis, disease, and treatment. Nevertheless, there is a need for additional biomarkers to improve prediction of impaired fertility, with emerging data suggesting that measures of ovarian reserve such as AMH may add value. Future studies will need to assess the risks of modern treatment regimens, including potential impact of targeted and immune-based therapies, and the role of ART on pregnancy rates in survivor populations. Recognizing the importance of survivorship issues, clinical trials should aim to incorporate patient-reported outcome measures to collect long-term fertility data. Understanding what is known and what is unknown about fertility risks is needed in order to counsel patients optimally regarding situations in which fertility preservation strategies may be needed and when patients can feel confident foregoing them. Newly diagnosed young patients with cancer and survivors may also benefit from the development and incorporation of counseling tools and guidelines for referral to oncofertility specialists.The authors have no funding sources to report.Affiliations of authors: Dana-Farber Cancer Institute ; Dana-Farber Cancer Institute and Boston Children’s Hospital ; Brigham and Women’s Hospital (ESG); Boston Children’s Hospital and Brigham and Women’s Hospital (MRL).


Strengthening routine immunization is one of the four prongs of the Global Polio Eradication Initiative. Achieving this requires improving immunization coverage in hard-to-reach areas. The objectives of this analysis were to assess levels of oral polio vaccination coverage and challenges in pastoral and semi-pastoral regions in Ethiopia. The analysis included vaccination-related data for children aged 12–23 months from the 2011 Ethiopian Demographic and Health Survey (EDHS) and from surveys carried out by the CORE Group Polio Project (CGPP) in 2013, 2015, and 2017. The EDHS data were from the entire regions (states) of Somali; Oromia; Southern Nations, Nationalities, and Peoples; Benshangul-Gumuz; and Gambella, whereas the CGPP data were for portions of these states where the CGPP was working and consisted entirely of pastoralist or semi-pastoralist populations. The overall polio immunization coverage rate showed upward trend from 39.6% in the 2011 EDHS to 72.6% for 2017 survey of children in the CGPP intervention areas. The evidence suggests that the CGPP was able to achieve increasing levels of coverage in the hardest-to-reach areas of these states and that the levels were higher than those achieved in the states as a whole. The strategies used by the CGPP/Ethiopia to increase coverage appear to have been effective. Other characteristics associated with full polio immunization included mother’s religion and education, whether the mother had heard about polio, knowledge on the effect of many polio vaccine doses, and age at first polio immunization. In the most recent Ethiopian Demographic and Health Survey (EDHS) published in 2016, only 30% of children living in rural areas in Ethiopia were fully vaccinated at the appropriate ages. Furthermore, only 53% of children in rural areas had received the full recommended three doses of oral polio vaccine (OPV 3).3Ethiopia’s last indigenous case of wild poliovirus occurred in 2001, and its last case of imported wild poliovirus was reported in 2014.2 The CGPP implements its programs through a network of international and national non-governmental organizations coordinated by a national secretariat.The CORE Group Polio Project (CGPP) has been active in Ethiopia since 2001, focusing on promotion of routine immunization services, polio immunization in particular, and surveillance for vaccine-preventable diseases in hard-to-reach pastoralist and semi-pastoralist populations and in high-risk border areas of Ethiopia in the following regional states: Somali; Oromia; Southern Nations, Nationalities, and Peoples (SNNP); Benshangul-Gumuz; and Gambella Region . A histo5).This article examines the population coverage of OPV in the CGPP program areas and the influences on coverage, using survey data collected between 2011 and 2017. The CGPP program areas contain 13 million children younger than 15 years, and the CGPP has trained more than 12,000 community volunteers (CVs) to promote immunizations and conduct surveillance in the CGPP program areas  survey and a consultant from the Faculty of Public Health at Jimma University for the baseline (2013) and final evaluation (2017) surveys. The data from the 2011 to 2016 EDHS survey used for the analysis were obtained from Measure DHS. The sampling procedure for the 2011 and 2016 EDHS survey is described elsewhere.The sampling procedure for the three CGPP surveys was identical: the standard WHO 30-cluster sampling methodology, but the sample size was expanded to compare differences between geographic areas within the CGPP program area. The sample size for specific zones within each region where the CGPP was working was determined by a probability-proportional-to-size (PPS) method. The sample sizes were as follows: 607 for the 2013 survey, 585 for the 2015 survey, and 672 for the 2017 survey.Only a subset of the 2011 and 2016 EDHS survey data was used for this study, namely, the data obtained from households in the regional states where the CGPP is operating: Somali, Oromia, SNNP, Benshangul-Gumuz, and Gambella and children aged 12–23 months. The CGPP implementation areas include five of nine zones of Somali, all three zones in Benshangul-Gumuz, all three zones in Gambella, two of 20 zones in Oromia, and two of 21 in SNNPR. All of these zones are located in pastoral and semi-pastoral regions that are considered hard-to-reach areas or in high-risk border areas .In all five surveys, data were collected from mothers during face-to-face household interviews. The respondents for the three CGPP surveys were the mothers or caregivers (hereafter referred to as mothers) of children 12–23 aged months, whereas the 2011 and 2016 EDHS survey collected childhood immunization data from mothers of children younger than 5 years. For our analysis, we extracted data of children aged 12–23 months from the EDHS surveys to match the analysis of CGPP survey category. Sociodemographic variables related to mothers and children were coded similarly across surveys before analysis. During the 2013 CGPP baseline survey, mother’s age, gender of child, birth date, place of birth, and OPV 2 status were not collected. Place of birth was also not collected in the 2015 survey. Thus, these data are missing from the tables in the results section.Oral polio vaccination coverage was calculated based on information written on the child’s vaccination card if present or, if not, on a report from the mother. For the calculation of OPV coverage, weights were used for the two EDHS surveys, whereas for CGPP surveys, no weight was used as the sample was carried out using PPS, but the standard errors for the cluster design were adjusted using the “svy” command in STATA. A child was considered fully vaccinated for polio if all three doses of OPV  were documented to have been received irrespective of whether the birth dose (OPV 0) had been received. Age at vaccination was determined for polio birth doses and for the first dose (OPV 1) provided that the date of birth and the date of vaccination were available. Those children who received OPV 0 within the first 2 weeks of birth and those who received OPV 1 at 6 weeks of age or later were considered timely vaccinated. In addition, the time intervals between OPV 1, OPV 2, and OPV 3 doses were calculated for those children whose dates of vaccination were recorded on their vaccination card. An interval of fewer than 28 days was considered as early vaccinated.We used STATA version 14.0  for analysis of the data and the chi-squared test of association between individual background characteristics and the five separate surveys. To assess the independent influence of specific background factors on full polio immunization, we used multivariable binary logistic regression and calculated adjusted odds ratios (ORs) and their associated 95% confidence intervals using only the data from the three CGPP surveys.Measure DHS gave permission for analysis of the 2011 and 2016 EDHS data. As the CGPP survey data were collected for monitoring and evaluation purposes rather than for research purposes, we did not request formal approval from an institutional review board to carry out the surveys in 2013, 2015, and 2017. Nonetheless, before the implementation of the surveys, the CGPP secretariat in Addis Ababa provided each of the zonal and regional health offices with an official letter stating the purpose of the surveys. We obtained verbal approval from all respondents after informing them that their participation was voluntary and that their responses would be kept strictly confidential.Supplemental Appendix Table 1.Supplemental Appendix Table 1).The other indicators were age at OPV 0 and OPV 1, and the interval between successive doses of OPV . Although OPV 0 is supposed to be provided within the first 15 days of life, 14.6% of children were given OPV 0 after 15 days of birth. There was improvement in this indicator over time, with a declining trend from 2011 (58.7%) to 2017 (9.5%) in the percentage of children in the CGPP program areas receiving their OPV 0 vaccination after 15 days of life. Similarly, 23.6% of children took OPV 1 before the minimum starting age of 6 weeks of age, with the highest percentage (28.2%) observed in 2015. In addition, the minimum interval between successive doses of OPV of at least 28 days was not respected in a substantial percentage of cases: the percentage of children who were vaccinated with OPV 2 less than 4 weeks after their vaccination with OPV 1 was 9.8% according to the dates shown in the vaccination cards in the two CGPP surveys (2015 and 2017), and 7.5% received OPV 3 before a sufficient time interval had elapsed after OPV 2 . Oral polio vaccine coverage levels are lower among mothers with no education compared with those with formal education, but coverage does not increase among those with at least some education as the educational level increases . Religion had a modest influence on OPV coverage. Mothers who are Christian have children with slightly higher coverage levels than do those who are Muslim or who are in the “Other” religious category .An analysis of the influences on OPV coverage indicate that coverage levels are slightly higher among mothers aged 20–29 years compared with younger and older mothers for the CGPP implementation areas .The influence of background characteristics on the knowledge of mothers about polio, on their opinion of the effect of many OPV doses on children, and on the age at first dose of OPV were assessed for the respondents in each of the three CORE Group surveys. As shown in About 92% of the respondents in the CGPP implementation areas had heard about polio and acute flaccid paralysis (AFP) in 2017 compared with 64% in 2013. Similar marked improvements in knowledge were noted between 2013 and 2015 and sustained in 2017 for awareness about the benefits of multiple OPV doses  and knowledge about when her child should receive its first polio vaccination (during the first 2 weeks of life) . During We fitted a multivariable binary logistic regression model for children who received the third polio vaccine dose (OPV 3), verified from card, to identify independent factors that influenced the uptake of OPV coverage, combining data from the three CGPP surveys  after considering the design effect of the cluster sampling. The important statistically significant independent influences on OPV 3 coverage, as shown in Our analysis included five data sets: the 2011 and 2016 EDHS survey , and subsequent surveys conducted by CORE Group in 2013, 2015, and 2017 in the pastoral and semi-pastoral hard-to-reach areas in Somali, Gambella, Benshangul-Gumuz, Oromia, and SNNPR where the CGPP was implementing its activities. The population coverage of polio vaccination was substantially higher in the CGPP implementation areas compared with the regions as a whole. The birth polio vaccine (OPV 0) coverage increased 3-fold and the coverage of the third polio vaccine dose (OPV 3) almost doubled from 2011 to 2017. Based on information from vaccination cards in the CGPP areas, the multivariate analysis demonstrated that OPV 3 coverage had a statistically significant associations with the date of the survey (coverage increased over time), mothers’ religion (higher coverage among children whose mothers were Christian), and education (higher coverage among children whose mothers had more education) as well as mothers’ who believe that multiple doses are beneficial and provide more protection.7 The objectives of this analysis were to assess levels of oral polio vaccination coverage and challenges in pastoral and semi-pastoral regions in Ethiopia.Strengthening routine immunization is one of the four prongs of the GPEI. The remaining road toward eradication will require improved access to populations in hard-to-reach areas.3 A study conducted in Jigjiga woreda, Somali Region, reported OPV 0 and OPV 3 coverage, based on card and history, of 14.6% and 44.1%,8 and the 2011 EDHS reported an OPV 3 coverage of 44.3%,6 lower than our findings for the CGPP implementation area. Arba Minch, in southern Ethiopia, reported a very high OPV 3 coverage of 86.3%.9Survey data from 2015 to 2017  showed oral polio vaccination coverage for the three doses  in pastoral and semi-pastoral region of Ethiopia was 74.2%; 17.0% had never received any OPV. Birth dose OPV (OPV 0) coverage for these areas estimated from the 2013, 2015, and 2017 surveys was 54.1%. The 2016 EDHS report indicated that for the national as a whole, 16% of children aged 12–23 months had not received any polio vaccinations, 81% had received their first dose of polio vaccine, and 56% received three doses of polio vaccine.3 whereas Kiptoo et al.10 found that lack of knowledge on immunization schedule, nomadic life style, low education level, and home delivery were significant factors associated with low immunization coverage in Kenya. Similarly, analysis of the Pakistan DHS 2012–2013 data identified maternal education as one of the factors significantly associated with complete OPV vaccination.11 A similar independent association between religion and vaccination status was reported in a study identifying factors associated with full immunization in Techiman Municipality, Ghana.12Full OPV coverage was significantly and independently associated with survey year, maternal education and religion, whether mothers had heard about polio, their knowledge about the effect of many OPV doses, and the starting age of first dose of polio vaccine. These results are consistent with other studies. The 2016 EDHS reported that vaccination coverage increases with mother’s education,13 another reason for low OPV 3 coverage might be the high dropout rate between OPV 1 and OPV 3. Although the national OPV dropout rate was substantial according to the 2011 EDHS (48.3%),6 in the CGPP implementation areas it was 13.7% or less in the 2013, 2015, and 2017 CGPP surveys. Notably, the average dropout rate over the period from 2013 to 2017 in the CGPP implementation areas was 7.3%, which is far lower than in the national 2011 and 2016 EDHS surveys.Although the high prevalence of home deliveries contributes to low OPV 0 coverage,10 and Angola,13 relatively higher dropout rates have been reported  can lead to a suboptimal immune response.15 The results of our study suggest that providers need to take more care in ensuring that polio immunizations are given at appropriate times.The vaccination series for OPV should be completed as early in life as possible as the age at highest risk of wild poliovirus infection in most developing countries occurs between 6 and 24 months of age. Currently, the recommended schedule for OPV is at birth, 6, 10, and 14 weeks of age. OPV administration at the recommended ages and in accordance with the recommended intervals between doses provides optimal protection.Although significant gains have been made in polio-related vaccination coverage in pastoral and semi-pastoral regions of Ethiopia, rates remained below the desirable coverage of 80–90%. Mothers’ knowledge about polio immunization, their religion, and educational level are important predictors of full polio vaccination status in their children. These findings point to the importance of educating young girls and women and ensuring access to primary and secondary schooling. Continuous polio awareness creation activities need to consider the local context and the involvement of local stakeholders, including community leaders and volunteers, and providing special training to CVs to provide birth dose OPV for home deliveries may be an area that might be put in place in hard-to-reach area in collaboration with community health workers. Polio-related education must include mothers with low or no education, as their children are at risk of incomplete vaccinations. Overall, almost all (85%) mothers in the CGPP surveys had heard of polio. However, more than a quarter had misconceptions about the benefits of more polio doses, and 48% wrongly stated the starting age for polio vaccine. Education should focus on increasing knowledge of vaccine benefits and the importance of timeliness in dosing.In addition, this study found that children of Muslim mothers were less likely to be vaccinated against polio. It is important to understand the factors that underlie this finding. The CGPP should continue to work with religious leaders, both Muslim and Christian, to dispel any misconceptions linked to religious teachings and strengthen accurate knowledge among Muslim mothers.Vaccination cards hold critical information about vaccination types, dates, and spacing. Without them, it is difficult to get accurate information. Survey data indicated that many caregivers did not have vaccination cards accessible or that they were possibly inaccurately filled out. The CGPP should continue to support parents in obtaining and retaining vaccination cards. Training and refresher training for health workers and immunization providers is necessary to improve quality and accuracy of recording. This is necessary to ensure that children are given needed vaccines and that they are given in a timely manner.Ethiopia has made great, notable strides in its efforts to increase polio vaccination coverage over the last 10 years. The CGPP has leveraged community networks and partnerships in the most hard-to-reach, highest risk areas of the country. These efforts have made it possible to increase polio vaccination coverage rates and to decrease dropout rates. Improving the timeliness of polio immunization administration requires continued emphasis.Supplemental materials


WIN 55,212‐2, disrupts recognition memory in mice. Here, we evaluate how prolonged, intermittent (30 days) exposure to WIN 55,212‐2 (1 mg/kg) alters recognition memory and impacts on brain metabolism and functional connectivity. We show that chronic, intermittent treatment with WIN 55,212‐2 disrupts recognition memory (Novel Object Recognition Test) without affecting locomotion and anxiety‐like behaviour (Open Field and Elevated Plus Maze). Through 14C‐2‐deoxyglucose functional brain imaging we show that chronic, intermittent WIN 55,212‐2 exposure induces hypometabolism in the hippocampal dorsal subiculum and in the mediodorsal nucleus of the thalamus, two brain regions directly involved in recognition memory. In addition, WIN 55,212‐2 exposure induces hypometabolism in the habenula with a contrasting hypermetabolism in the globus pallidus. Through the application of the Partial Least Squares Regression (PLSR) algorithm to the brain imaging data, we observed that prolonged WIN 55,212‐2 administration alters functional connectivity in brain networks that underlie recognition memory, including that between the hippocampus and prefrontal cortex, the thalamus and prefrontal cortex, and between the hippocampus and the perirhinal cortex. In addition, our results support disturbed lateral habenula and serotonin system functional connectivity following WIN 55,212‐2 exposure. Overall, this study provides new insight into the functional mechanisms underlying the impact of chronic cannabinoid exposure on memory and highlights the serotonin system as a particularly vulnerable target.Elucidating how cannabinoids affect brain function is instrumental for the development of therapeutic tools aiming to mitigate ‘on target’ side effects of cannabinoid‐based therapies. A single treatment with the cannabinoid receptor agonist,  We found out that adult mice chronically exposed to WIN 55,212‐2 displayed impaired recognition memory and differences in metabolic brain activity and dysfunctional connectivity in circuits that underlie memory processing, thus providing new insights into the functional mechanisms that underlie the impact of chronic cannabinoid exposure on memory.This work was designed to elucidate the impact of chronic, intermittent cannabinoid exposure on brain metabolism, functional brain connectivity and recognition memory. We carry out three different analysis: first, we evaluated recognition memory in the Novel Object Recognition Test (NORT); secondly, we studied brain metabolic activity in these animals using ad libitum access to food and water. Animal behaviour experiments were conducted during the light phase at around the same time each day (10 : 00). All experimentation followed the European Community Guidelines (Directive 2010/63/EU) and the Portuguese law (DL 113/2013) for Animal Care for Research Purposes, and were approved by the ‘Instituto de Medicina Molecular’ Internal Committee and the Portuguese Animal Ethics Committee – Direcção Geral de Veterinária. The experimental protocol was not preregistered. Animals were habituated to the presence of the investigator and handled for 5 days before testing. A total of 40 mice were used, 20 were treated with WIN 55,212‐2 and 20 were receiving the vehicle only. All animals were used for behaviour analysis, which was performed in two series of 10 mice per drug condition, giving a total of 20 mice per condition. For connectivity analysis, 10 mice per condition were used. To determine whether an animal would be allocated to the control or experimental treatment group a pseudo‐randomization procedure was employed. Animals were tagged and distributed in groups of five animals to each housing cage . Subsequently, four cages were randomly attributed to each treatment condition . To do so, a number was attributed to each cage and randomly drawn. Drawn cage numbers were distributed sequentially to either the control or the experimental group .Adult (8–12 weeks old) male C57BL/6 mice   were used. Animals were housed in a temperature (22/24°C) and humidity (45–65%) regulated room with a 14/10‐h light/dark cycle (07:00–21:00) with i.p.) injections was < 0.6 μL per mouse, and control animals were injected with equivalent amounts of vehicle (2 mL/kg).WIN 55,212‐2  was suspended in dimethylsulfoxide  before injection. The amount of dimethylsulfoxide present in the solutions used for intraperitoneal . To refine the results obtained by software measures, a post‐analysis was conducted. In the post‐analysis, the investigator was blind to the experimental condition. Exploratory behaviour was quantified as the amount of time (seconds) animals spent investigating each object . The number of approaches that included sniffing the object, rearing towards the object or touching the object was counted .NORT was conducted in a wooden square open field arena (40 × 40 × 40 cm) as previously reported . The reference point used by the software to determine the position of the animal was the centre of the mouse dorsum, as done previously in our Institute . Environmental conditions and animal manipulation procedures were kept as similar as possible between animals.Anxiety‐like and locomotor behaviour were analysed in the OFT and EPM. Behaviour in the OFT was analysed before the NORT. The OFT was used to assess individual behaviour when animals were placed in a novel environment , in accordance with previously published protocols . After the injection animals were returned to their home cages. Forty‐five minutes after isotope injection, animals were killed by cervical dislocation and subsequently decapitated. A terminal blood sample was collected from the neck by torso inversion, to determine circulating glucose levels (Accu‐Chek Aviva Blood Glucose Monitor). The brain was dissected out and immediately frozen in isopentane (−40°C) and stored at −80°C until sectioning. Blood samples were centrifuged to separate the plasma for determination of plasma 2‐deoxy‐D‐[14C]‐glucose concentration by liquid scintillation analysis (Table S1).Local cerebral glucose utilization (LCGU) was determined after the final WIN 55,212‐2 (or vehicle) treatment  to high‐resolution autoradiographic film  for 7 days, after which they were developed in accordance with the manufacturer's instructions. Autoradiographic images were analysed by computer‐based images analysis (MCID/M5) (RRID:SCR_014278). The local isotope concentration for each brain region of interest (RoI) was obtained directly from the optical density of autoradiographic images relative to that of co‐exposed 14C‐standards. Forty‐nine anatomically distinct RoI were measured with reference to a stereotactic mouse brain atlas . The whole brain average 14C concentration was determined as the average 14C concentration across all sections in which a RoI was measured. No animals were excluded from this data set.Brains were coronally sectioned in a cryostat (−20°C) at 20 μm. Three consecutive sections were retained from every 60 μm and dried rapidly onto slide covers on a hot plate (70°C). Autoradiograms were obtained by placing these sections, along with pre‐calibrated https://CRAN.R-project.org/package=pls, Mevik and Wehrens 14C‐2‐DG imaging data was undertaken as previously reported , or significantly lost or newly gained functional connectivity .Regional functional connectivity was analysed in control  and WIN 55,212‐2‐treated mice. To elucidate the impact of chronic WIN 55,212‐2 exposure on regional functional connectivity, we applied the Partial Least Squares Regression (PLSR) algorithm . A test for outliers was performed using the Graphpad Outlier Calculator. NORT data were analysed using two‐tailed, paired or unpaired Student's t‐tests, as appropriate for each condition and as indicated in the figure legends. Overt alterations in LCGU were statistically analysed by Student's t‐test, with discrete RoI treated as independent variables, as previously justified  or R , WIN 22,515‐2‐treated animals did not . A similar effect was seen when analysing the novelty preference index (NPI) with control animals showing a significant preference for the novel object during the test phase , while WIN 55,212‐2‐treated animals did not  and animals treated chronically with WIN 55,212‐2 explored the two identical objects for a similar amount of time . By contrast, WIN 55,212‐2‐treated animals showed significant hypermetabolism in only one of the brain regions analysed, the Globus Pallidus (GP) . These results support altered function in the basal ganglia–thalamic–hippocampal circuits . Blood glucose and level of 14C‐2‐DG in the plasma were not significantly altered in control and WIN 55,212‐2‐treated animals (Table S1).Chronic, intermittent WIN 55,212‐2 administration induced both increases and decreases in LCGU on a brain region‐dependent basis. WIN 55,212‐2‐treated mice showed marked hypometabolism in the dorsal subiculum of the ventral hippocampus (DSub), the thalamic mediodorsal nucleus (MD) and in the habenula (Hab) . Analysis by means of PLSR algorithm revealed significant modifications in functional connectivity in the mice that had received WIN 55,212‐2 . In animals treated with WIN 55,212‐2, functional connectivity of the DSub was significantly altered. Specifically, significant decreases in  and lost  connectivity with multiple PFC regions were seen in animals treated chronically with WIN 55,212‐2. By contrast, chronic WIN 55,212‐2 administration resulted in new abnormal functional connectivity between the DSub of the ventral hippocampus and subfields of the dorsal hippocampus . In addition, functional connectivity of the DSub to other subfields in the ventral hippocampus was significantly enhanced  or gained (DG) in WIN 55,212‐2‐treated animals. This suggests that the local connectivity of the ventral hippocampus DSub to other hippocampal subfields was significantly increased as a result of chronic WIN 55,212‐2 administration. In addition, functional connectivity of the DSub to the perirhinal cortex was abnormally gained in response to chronic WIN 55,212‐2 treatment. Furthermore, the DSub gained new functional connectivity to discrete thalamic nuclei  and to the serotonergic MR following chronic WIN‐55,212‐2 administration.In control animals the Hab was functionally connected with several PFC subfields and several striatal, hippocampal and amygdala regions (Table S4). Following chronic WIN 55,212‐2 treatment the functional connectivity of the Hab was significantly altered. On one hand, the Hab lost functional connectivity with multiple PFC subfields  and showed significantly lost  and decreased connectivity  to several regions of the basal ganglia. By contrast, WIN 55,212‐2 treatment provoked abnormal gains in connectivity between the Hab and subfields of the nucleus accumbens , with the serotonergic system .In control animals the MD thalamic nucleus was significantly functionally connected to multiple subfields of the prefrontal cortex (PFC), other cortical regions and other thalamic nuclei (Table S5). The functional connectivity of the MD was significantly altered in mice treated chronically with WIN 55,212‐2. This included significantly lost  or decreased  connectivity to other thalamic nuclei, and cortical regions . By contrast, chronic WIN 55,212‐2 treatment resulted in new, abnormal functional connectivity between the MD and multiple subfields of the septum/diagonal band of Broca .Finally, in control animals the GP was functionally connected to numerous regions of the septum/diagonal band of Broca, and to striatal and cortical regions (Table S6). As a consequence of chronic WIN 55,212‐2 treatment the functional connectivity of the GP was significantly modified. The GP significantly lost functional connectivity with several regions of the septum/diagonal band of Broca  and to the striatum . By contrast, the GP gained abnormal new connectivity with PFC  and to the serotonergic raphe (MR and DR) following chronic, intermittent WIN 55,212‐2 treatment.In this work we demonstrated that mice chronically exposed to the non‐selective cannabinoid receptor agonist, WIN 55,212‐2, displayed disrupted cerebral metabolism and abnormal functional connectivity in the cortico–thalamic–hippocampal circuits that underlie recognition memory. This includes compromised perirhinal–hippocampus–prefrontal cortex and thalamo‐prefrontal functional connectivity. In parallel, we observed deficits in recognition memory as a consequence of chronic WIN 55,212‐2 administration without signs of altered motor abilities and anxiety‐like behaviour.1Rs on synapses inhibit glutamatergic and GABAergic transmission, modulate different forms of synaptic plasticity and control neural oscillations that support behaviour and diverse cognitive functions, including learning and memory  and the mediodorsal nucleus of the thalamus  thalamic nuclei as a consequence of WIN 55,212‐2 administration, which could also contribute to the deficits in recognition memory seen in these animals. Indeed, there is building evidence supporting a cortico–thalamic–hippocampal network, including the MD, that underlies recognition memory, in part as a result of the ability of the mediodorsal thalamic nuclei to act as a relay between the perirhinal and the medial prefrontal cortex  abolished the impact of acute WIN 55,212‐2 administration on recognition memory, which was evaluated with the same NORT paradigm and using mice of the same age as used in this study  from PORTUGAL 2020 and Fundação para a Ciência e Tecnologia (FCT), by an FCT project  and by a Twinning action (SynaNet) from the EU H2020 Programme (project number: 692340), which covered short‐term scientific missions of FMM at ND laboratory and of ND at AMS laboratory. FMM was in receipt of SFRH/BD/89582/2012 FTC fellowship. AMS is a handling editor for the Journal of Neurochemistry.All experiments were conducted in compliance with the ARRIVE guidelines.Table S1. Body weight and blood parameters for animals undergoing the 14C‐2‐DG experiment.Table S2. Local cerebral glucose utilization (LCGU) in WIN 55,212‐2 and control animals.Table S3. Functional Connectivity of the Ventral Hippocampal Dorsal Subiculum.Table S4. Functional Connectivity of the Habenula.Table S5. Functional Connectivity of the Mediodorsal Thalamic Nucleus.Table S6. Functional Connectivity of the Globus Pallidus.Click here for additional data file.