The COVID-19 pandemic has caused an unprecedented global public health emergency. Vaccine uptake in low and middle income countries (LMICs) lags developing nations and immunity from vector-based vaccines commonly used in LMICs may be inferior to mRNA vaccines. Thus, defining clinical characteristics that can help identify and triage cases and allocate resources in LMICs of priority. Hyperglycemia has been associated with higher morbidity and mortality in numerous diseases and in critical illness. We seek to understand the relationship between COVID-19 and hyperglycemia.This is a single center retrospective review of cases with COVID-19 between January 2021 and June 2021. Adult patients >18 years of age were reviewed and those with a molecular-based laboratory confirmed SARS-CoV-2 infection were included in our study. Patients with known diabetes, elevated A1C or prior steroid use within 2 weeks of admission were excluded. Clinical characteristics, demographics, glucose levels, C-reactive protein (CRP) and ferritin were reviewed.A total of 120 patients were reviewed, of which 60.8% were male. Hyperglycemia ( >140mg/dL) was present in 57.5%. Hyperglycemia was associated with elevation of inflammatory markers including CRP and Ferritin (p=0.12) (Table 1). Hyperglycemia was more common in patients requiring supplemental low flow oxygen (table 2) and was more common in patients who did not survive . The mortality rate was higher in the hyperglycemia group with 61.5%, a statistically significant finding.Association between hyperglycemia and inflammatory markers.Supplemental oxygen needs and hyperglycemia.Survival and hyperglycemia.Hyperglycemia on admission was an independent risk factor for disease progression and death. Inflammatory markers were also higher in patients with hyperglycemia. These patients had no prior steroid use or diabetes. Thus, it is possible that it reflects inflammation, stress, or endocrine end-organ damage due to SARS-CoV-2. If validated in larger studies, this simple test can help clinicians identify patients at risk of decompensation and allocate resources and therapeutics accordingly.All Authors: No reported disclosures.


The mortality rate of people with neurological disorders, such as stroke, Parkinson’s disease, spinal cord injury, cerebral palsy, multiple sclerosis is decreasing due to advancements in medical research, as mentioned for example in . HoweverThis Research Topic aims at presenting innovative robotic solutions to boost the rehabilitation process for neurological disorders. This Issue highlight recent advances in the development of these robotic devices including a comprehensive review of recent developments in the area of rehabilitation robotics, information, and recent achievements on both therapeutic and assistive robots, focuses on the state-of-the-art and representative advancements in the design, simulation, control, analysis, implementation, serious games, and validation of rehabilitation robotic systems.This Research Topic contains feature articles as focused on theoretical and experimental results dealing with techniques for the design, simulation, and control systems for rehabilitation devices for upper and lower limbs including serious games and virtual reality. The presented solutions explore the development of devices that are cost-effective with high impact and potential for future developments to clinical applications. Still the Research Topic will require careful research attention in the near future for progressing human wealth.Alves et al.), the authors outline the development of serious games strategies used together with a cable-driven robot applied for bimanual rehabilitation. Cable-driven robots can be an ideal fit for performing post-stroke rehabilitation due to their specific features in function have small and lightweight moving parts and a relatively large workspace. The results demonstrate the engineering feasibility and effectiveness of the proposed cable-driven robot as a valuable tool to augment the conventional physiotherapy protocols and for providing reliable measurements of the patient’s rehabilitation performance and progress. The second article  presents a design and prototype of an underactuated hand exoskeleton with fingers coupled by a gear-based differential. Exoskeletons devices represent a promising opportunity for rehabilitation and assistance to people presenting neurological diseases. However, there are still some limits to the diffusion of robotic technologies for neurorehabilitation, notwithstanding their technological developments and evidence of clinical effectiveness. In the article , the authors present an anti-disturbance sliding mode control for a novel variable stiffness actuator to be applied for lower limb rehabilitation. Lower limb exoskeletons are widely used for rehabilitation training of patients suffering from neurological disorders. In this paper, a novel load-adaptive variable stiffness actuator is used to design an ankle exoskeleton with a sliding mode controller based on a disturbance observer. The simulation and experimental verification are performed, and the wearing experiment confirms that the controller can realize zero-impedance control of the designed ankle exoskeleton. The design of a suspension lever mechanism with elastic elements for a robotic rehabilitation system for the lower limbs was presented in the fourth article, . The article discusses the design of a suspended lever mechanism with elastic elements, which is used as a safety device in a robotic system for the rehabilitation of the lower limbs. The article analyzes the existing mechanical structures of devices for rehabilitation, identifies the problems of operation, design, and safety systems, and suggests a modern design for the device. The article addresses the issues present in the current mechanical designs with a brief discussion on the system architecture. The author of the last article  presented a systematic review and meta-analysis of the effect of virtual reality on balance function in children with cerebral palsy. Virtual reality therapy is popular in treating children with cerebral palsy as a modern technology for rehabilitation. The systematic review and meta-analysis included all randomized controlled trials that included children with cerebral palsy. Results showed that the virtual reality intervention was beneficial for balance, however, cautious implementation is needed in clinical applications.In a first article, (


However, the process of admitting patients to inpatient rehabilitation is a complex process. This is yet to be standardised across the European Union, as the approach to geriatric rehabilitation tends to vary from one Member State to another. (2) Objective: To discuss evidence-based practice with clinical experts, in order to define geriatric rehabilitation and admission criteria based on the Maltese population. (3) Method: The study entailed conducting four panel sessions using a purposive sample of thirteen local clinicians with extensive knowledge in clinical rehabilitation and healthcare management. A total of 48 items, based on the literature and clinical experience, were presented to the panel. Data analysis was done quantitatively and qualitatively, using IBM SPSS Statistics Version 24 and thematic analysis. (4) Results: The panel formulated a definition of rehabilitation, which shared common elements with the definition provided by the World Health Organization (WHO) and other sources/literature. The panel agreed on a list of eight criteria for appropriate inpatient geriatric rehabilitation admission in Malta. Consensus was also reached on: the need for a consultant-led multidisciplinary approach to assessment; the adoption of a standardised assessment processes for an equitable chance for all older adults assessed; the benefit of digital health in assessments; and the consideration that most patients would have some form of rehabilitation potential, depending on availability of resources. (5) Conclusion: Inpatient geriatric rehabilitation hospitals should have a unified strategy for rehabilitation services. The conclusions reached by the panel, could be useful in supporting the clinical evidence and establishing future rehabilitation guidelines and standards for inpatient rehabilitation. An ageing population is inevitably linked to increasing rates of morbidity and disability. By the end of 2020, 18.9% of Malta’s total population was aged 65 years and over [Malta’s only state-run rehabilitation hospital admits, among other patients, older adults for inpatient rehabilitation. Since this hospital has a limited number of inpatient beds, appropriate admission criteria would be essential in ensuring that truly eligible patients are admitted.Rehabilitation consultants and geriatric consultants, who assess patients for admission to inpatient rehabilitation, make daily clinical decisions on patients’ rehabilitation potential and eligibility for inpatient admission. There is currently neither an established systematic approach to assessing patients for rehabilitation potential nor a clinical guide for rehabilitation admission criteria ,3,4. ResIn view of the above, the aim of this study was to pitch published evidence-based practices to the panellists and for them to discuss it, in order to define GR and admission criteria with reference to the Maltese population. Through the use of the Delphi method, we sought to involve a panel of national experts towards reaching a consensus on fundamental principles in GR.The Delphi method is a planned procedure that uses rounds of questions to gather experts’ opinions, without requiring face-to-face meetings . The resA set of criteria was established by the study team to identify the ideal experts to form part of the Delphi panel, taking into account the criteria required when forming a panel ,15. In oThe research team identified thirteen experts through purposive sampling. These were individually contacted by e-mail and, upon expressing their interest in participating in the study, were provided with an information sheet and a consent form, which provided a detailed explanation of the aims of the study and what their involvement entailed. All of the selected experts that were invited consented to participate in the study. The panel of experts consisted of MDT professionals working in inpatient rehabilitation, namely: (1) geriatricians and rehabilitation consultants; (2) allied health professionals, including physiotherapists and occupational therapists; (3) nurses; and (4) social workers, all in management positions in rehabilitation, including that of older adults. These experts all worked on a full-time basis within the same Maltese rehabilitation hospital and had been fulfilling the same role for a minimum of five years.In Malta’s rehabilitation hospital, members from the multidisciplinary team hold weekly meetings to discuss individual patient cases, progress, and plans for rehabilitation. These meetings are geriatric-consultant-led, and include the nurse, physiotherapist, occupational therapist, and social worker. Therefore, realistically, the panellists chosen for the study consisted of these professionals. This made the findings more contextually relevant.SurveyMonkey, an online-survey platform, was used for all the rounds of Delphi. The surveys planned were sent individually to each panel expert, through an e-mail link. The participants were given one week to submit their responses; any panellist who had not yet filled in the questionnaires received a reminder in the week following the set deadline. Maintenance of participation levels between 50 and 80% was necessary to ensure that a valid consensus could be reached ,17. In oThe aim of the Delphi survey was to define geriatric rehabilitation (GR), discuss admission criteria for inpatient GR, discuss the composition of the MDT, and discuss the use of digital technology to help guide clinicians to make consistent, transparent decisions on GR admission, for the Maltese population. The statements presented in the rounds were mostly based on evidence regarding rehabilitation and GR potential, and on responses given by the panellists from previous rounds.The first round of Delphi, served to create a baseline for the panel, in order to ensure that the points for discussion and the theme of the study were clear and concise ,14. PartThe first round concerned: rehabilitation definitions; domains that affect RP; admission criteria; assessment measures; and outcomes of rehabilitation—all of which were initially compiled from the literature. This was an introduction that served as the foundation of the questions in the remaining rounds. Indeed, the second questionnaire was created using the experts’ responses from the first Delphi round, eliminating the items that had already attained a high level of homogeneity, and maintaining the ones that had not shown a sufficient level of consensus. The ideas generated by the experts in their remarks from the first round were taken into consideration when these elements were revised and reintroduced to the panel in the second round. New multiple-choice questions were also included. A total of 48 items were presented to the panel during the rounds. Consensus was considered to have been achieved if at least 70% of the panellists responded to the same statement from among a choice of ‘strongly agree’ and ‘agree’, or ‘strongly disagree’ and ‘disagree’ ,11. ThisOn the other hand, qualitative data analysis was carried out manually. Each question was evaluated thoroughly, and the main points and common themes were grouped together. This process is called thematic analysis, as described by Bryman .This study was reviewed by, and received approval from, the University of Malta Research Ethics Committee. Each panellist was given one code which they were required to apply when completing the rounds. A second code was allocated to each participant, which was only known by the researcher . This coThe results of the rounds are presented in sections. The first section  providesThe panel consisted of thirteen experts—53.8% male and 46.2% female—with 38.5% aged between 45 and 54 years; 46.2% held a master’s degree, while 46.2% worked in rehabilitation for over 20 years, across clinical and managerial roles. All experts were Maltese nationals. Further demographic and educational characteristics of the participants is presented in The first round involved the circulation of two questionnaires, one aimed at collecting the experts’ demographics and one covering questions regarding the topic under discussion. The latter was composed of eight questions, one of which was quantitative in nature. Most questions showed similarities and convergence, and the aim of this round was to generate ideas and link the responses to the literature . Most reThe second round consisted of six questions, one of which was open-ended. The first three questions were reproduced from the first round. The remaining three questions were first introduced in this round. The participants were required to respond to more defined statements regarding definitions of rehabilitation, which domains have the highest implication on rehabilitation, and which outcome measures should be used to assess these domains. The round also considered which clinician/s from the multidisciplinary team (MDT) would be the most suitable for conducting the assessment, and if digital technology could aid this assessment process in any way.Percentage consensus was calculated, with three questions showing convergence, and hence were eliminated from subsequent rounds. Three questions were reintroduced in the following round, even though there were similarities in the responses. These similarities emerged through the ‘additional comment’ field following the questions, as some panellists clarified the statements that merited further discussion and thus offering higher convergence.The questionnaire in the third round consisted of ten questions, which included sub-questions to a total of twenty-one items. This round sought to gather the panel’s views on the MDT involvement in assessment, the admission criteria, and the use of digital technology in the assessment process. The questions that were reformulated from the second round were intended to narrow down the responses, aiming for a more concise and homogenous answer from the panel.A good degree of convergence was achieved in three of the five questions asked on the MDT approach to assessment, in three of the four questions asked on digital technology use in assessment, and in five of the twelve questions asked on admission criteria to inpatient rehabilitation. This meant that the following round required further clarification and reformulation of statements regarding admission criteria.The questionnaire in the fourth round consisted of four questions, with sub-questions to a total of thirteen items discussed. These included re-submitted questions on the MDT assessment process and on digital technology involvement, and also re-introduced and new questions on admission criteria to inpatient rehabilitation. The aim of this round was to continue evaluating the panel’s views on which patients should be considered eligible for inpatient rehabilitation, and which would benefit from other services instead.Convergence was noted in most replies. However, four items did not deliver a homogenous reply and the responses were distributed throughout the five-point Likert scale, and thus were not conducive to reaching a final conclusion. This was noted in statements that did not reach consensus in the previous round, and not in new statements introduced. For this reason, and since this was the final round of the Delphi survey, these items were excluded from the list of appropriate admission criteria. A summary of the survey results was sent to the panel. A total of 20.8% of the questions asked were open-ended in nature. The main themes were extracted, and the statements were formulated and presented to the panel for discussion. Any themes that obtained immediate consensus were eliminated from subsequent rounds.In the first round, the participants were presented with standard definitions of rehabilitation, geriatric rehabilitation (GR), and rehabilitation potential (RP), and were asked to comment on the statements. A total of 84.6% agreed with these definitions but added to the description of rehabilitation potential. The remaining 15.4% maintained that there should be no distinction between rehabilitation and GR. The results were collated together and one statement to define rehabilitation was sought and presented to the panel in the second round, which subsequently obtained a 92.3% consensus.The panellists were asked about domains that affect RP and the open-ended follow-up question was to ‘list any other factors that affect rehabilitation and a patient’s potential’. A total of 76.9% maintained that the patient’s character, motivation, and willingness to undergo rehabilitation would play a key role in the patient’s performance, and thus the outcome. The comments also included the mental state of the patient, including depression and anxiety, and the ability to participate in, and benefit from, rehabilitation.Three questions achieved consensus in the first round, which aided in the formulation of the admission criteria statements that were presented in Rounds 3 and 4. The responses obtained in the close-ended questions were compared to these results, and are presented in the following section.In contrast to the above, two questions did not reach consensus during the first presentation of the open-ended question, and required clarification and re-discussion in the ensuing round. These questions concerned which outcome measures should be used when assessing patients for GR inpatient admission. There were a number of outcome measures mentioned in this section, none of which were mentioned by the majority. In total, 34 outcome measures were mentioned, with the most commonly mentioned being the Barthel index or BI (14.7%), the functional independence measure or FIM (14.7%), the mini mental state examination or MMSE (11.8%), the clinical frailty scale or CFS (11.8%), and the Montreal cognitive assessment or MoCA (8.8%). The responses obtained were compared to the existing literature  and a liThe remaining two questions were free-text questions, which allowed the panellists to comment freely on anything that was discussed during the panel sessions. These were asked during Rounds 3 and 4. The panellists commented on various topics of the survey, including the benefits of a standardised approach to patient assessment, the benefits and risks of using digital technology to compile and store assessment data, and the challenges faced by clinicians when trying to identify the most adequate admission criteria. In these two questions, none of the themes extracted showed convergence. Hence, these responses were used as a basis for further statements or to enhance the robustness of already discussed statements.Notwithstanding the above, similarity was noted within the panel in mentioning that GR assessment should be patient specific, and depending on individual characteristics, patient motivation, and rehabilitation potential. In view of clinical complexity and the frailty of referred patients, the panel questioned the ability of clinicians to predict rehabilitation outcomes in an effective manner, in the absence of a standardised approach to assessment, or the presence of only one clinician, assessing the patient and making the decision alone.A total of 79.2% of the statements were close-ended in nature. Convergence was noted in 52.6% of the statements, more specifically: 2.6% on rehabilitation definitions and potential; 7.9% on outcome measures and domains; 10.5% on composition of the MDT; 18.4% on statements on admission criteria; and 13.2% on the use of digital technology in the assessment process.The panellists were presented with a list of domains, extracted from the existing literature , and werThe MDT involvement in patient assessment constituted 15.8% of the statements, of which 10.5% reached convergence. The panel agreed that the admitting team should consist of a consultant geriatrician or rehabilitation specialist, a physiotherapist, an occupational therapist, and a social worker, with the potential addition of a speech and language pathologist, psychologist, dietician, and other pertinent professionals, depending on the individual patient’s needs. The panel responses are shown in The remaining 5.3% of the MDT statements showed a lack of agreement on the involvement of nurses. The panel was asked whether nurses should be involved in GR patient assessment, with 46.2% of the panel being in agreement. Since consensus was not reached, the statement was represented to the panel in the following round, noting an increase to 61.6% agreement. The panel showed conflicting responses that the nurse should be involved mostly in cases where the patient would require specialist nursing, such as wound management or pain management, on a referral basis.The panellists were asked what they considered to be a ‘good’ outcome of rehabilitation and a ‘poor’ rehabilitation potential. A total 46.2% of the panel maintained that a good outcome of rehabilitation would occur when the patient could be reintegrated within the community, in previous housing or living with a family member, while 30.8% considered a patient reaching their maximal potential as a good outcome. Another 30.8% mentioned reaching patient and caregiver’s goals and objectives of rehabilitation. In contrast, most panellists (84.6%) considered poor prognosis and multiple comorbidities to be the lead determinants of poor RP. The panel argued that decreased medical stability tended to lead to a decrease in ability to co-operate during therapy sessions, thus rendering rehabilitation more challenging. Co-operation would be further decreased in the presence of impaired cognitive function, as mentioned by most panellists (61.5%), and possibly leading to a lack of willingness and motivation to adhere to treatment sessions. A lack of social support was considered by 38.5% of the panellists, along with 15.4% believing that the level of independence prior to the event that led to hospitalisation would have an impact on the success of the rehabilitation phase.Based on the above-mentioned responses, the panellists were then asked to discuss which admission criteria they deemed necessary for inpatient GR. Two particular statements represented a higher disagreement level, yet did not reach dis-consensus. These statements addressed the medical stability of the patient and read as follows: ‘Has a need for daily nursing and medical care’ and ‘Multiple admissions in the previous year’. Therefore, by not reaching a suitable level of agreement, the panel shifted towards admitting patients who would be sufficiently medically stable to withstand intensive rehabilitation programmes.Another statement that remained inconclusive concerned the cognitive status of the patient. There was no agreement with the statement that patients with moderate-to-severe cognitive impairment should be excluded from GR. While some panellists maintained that patients with cognitive impairment could still be rehabilitated if the staff would be well trained and the environment adequate in accommodating their needs, others maintained that if the cognitive impairment were to be a hindrance to rehabilitation compliance, or the impairment should cause a challenge for the relatives if the patient were to be rehabilitated, then the patient should be excluded from inpatient GR. Delirium and premorbid cognitive status were also mentioned as important considerations for GR admission. Furthermore, panellists mentioned the importance of assessing cognitive status to be in the best position to plan treatment sessions accordingly.The panellists were also presented with questions regarding the use of digital technology in the process of patient assessment for admission to GR. The panel agreed that a digital standardised assessment should not be the final and definite decision that would replace clinical judgement. Nevertheless, it should serve as an assistive tool that would ensure that the patient be assessed in a holistic manner, identifying patient needs and expectations, and supporting the better planning of treatment and rehabilitation care. A total of 15.4% of the panel highlighted the point that a standardised digital approach would offer greater transparency in the assessment process, and would allow increased ease of communication with other healthcare entities and professionals, as well as the patients themselves and their caregivers.This study was the first in Malta to employ the Delphi method in seeking to identify what is understood by rehabilitation and which admission criteria are the most appropriate for inpatient geriatric rehabilitation (GR). There was a 100% response rate in all four rounds, with all the panellists replying to the questionnaires within the set time frame. Consensus was not reached on: the nurses’ involvement in inpatient admission assessments and on admission criteria concerning patient cognitive level; previous long-term care applications; the need for daily nursing care; and the number of admissions to hospital in the previous year.Having stated the above, similarities were noted in the responses, even when the panel chose ‘neutral’ in their scale response. This suggests that there were implied shared criteria of acceptability among the panellists, despite there being no current standardisation of assessment. By the end of the panel sessions, the experts agreed on a list of eight criteria for appropriate inpatient GR admission . AdmissiThe definition of rehabilitation that was agreed upon among the panellists was as follows: ‘Rehabilitation is a multidisciplinary approach of interventions, aimed to maximise functional independence and facilitate psychosocial adjustments to residual disability, to allow a patient to function successfully in the community’. This was similar to the definition offered by the WHO  and thatIn the current study, it was ultimately agreed that GR assessment was an extensive and detailed process that would require a multidisciplinary approach. This would help structure a patient-oriented rehabilitation plan. Inpatient GR services should evaluate the assessment process on a regular basis, to detect any flaws, or areas of improvement. This could be based on evaluation of: duration of stays in hospital; rates of mortality during rehabilitation; fluctuating numbers of admissions and patients referred for long-term care institutions; and functional abilities in the community with follow-ups . The assIt appeared that some of the experts on the panel expressed their views not solely based on their clinical expertise and knowledge, but were also influenced by the administrative and managerial problems encountered in the day-to-day running of rehabilitation in Malta. The panel indicated that the assessment process should be more standardised and thorough, portraying some similarities with the comprehensive geriatric assessment (CGA) model ,22,23. HAlthough the majority of the panel maintained that RP and admission criteria should be based on improving the patient’s functional abilities, a few panellists also mentioned the importance of offering rehabilitation even in cases requiring higher levels of medical, adaptive, or palliative care ,25. FurtThe panel did not reach consensus on admission criteria regarding the cognitive level of the patient. The panellists had conflicting responses as to whether patients with dementia or cognitive impairment tended to have lower RP and lower compliance rates to treatment, as was noted in at least one study . In contFinally, a number of panellists also mentioned that no patient should be denied rehabilitation, and that patients should be equally eligible for inpatient GR, irrespective of their premorbid or current condition. Nevertheless, it was also pinpointed that the available resources presented ethical challenges and the need for prioritisation of service allocation. This may be viewed as a contradiction to the comments provided above.It should be noted that the use of the Delphi method approach is not intended as an alternative to rigorous scientific research, but to inform it ,13. ConsSample size recommendations varied across literature, with minimum numbers including five, ten, or twelve panellists to be sufficient to enable consensus ,28,29. TThe anonymity and confidentiality employed during the rounds helped minimise the bias in the panel responses, which may have otherwise been influenced by existing relationships between the panel and the researchers involved. The Delphi method reduced the possibility of any panel members being reluctant to express their views if these contradicted others or if some panel members were more expressive than others. Existing literature has also highlighted this advantage of the Delphi method ,14,17.This study proposes that geriatric rehabilitation (GR) and its assessment is a complex decision-making process through which clinicians are required to make clinical judgements on the patient’s potential and expected outcomes if admitted to an inpatient GR programme.The panel agreed about the need for a consultant-led multidisciplinary approach to rehabilitation assessment and management. The inclusion of other stakeholders, such as the patients themselves and their caregivers, could offer a wider view of rehabilitation in older adults. This research focused solely on clinicians working in the field of rehabilitation, but a further step could be taken to include these stakeholders in future research. The panel believed that, while all patients had some form of rehabilitation potential, the provision of rehabilitation would depend on the wishes of the patients and caregivers, and on the resources available.The conclusions obtained by this panel could be useful in supporting the existing clinical evidence and establishing rehabilitation guidelines and standards for inpatient rehabilitation. Inpatient GR hospitals should have a unified strategy for rehabilitation services. Considering that the Delphi panel findings represent local expert opinions, rather than more far-reaching scientific evidence, further research to solidify the conclusions would be crucial . Neverth


Steroid hormones are capable of diffusing through cell membranes to bind with intracellular receptors to regulate numerous physiological processes. Three classes of steroid hormones, namely androgens, estrogens and glucocorticoids, contribute to the development of the reproductive system and the maintenance of fertility. During the past 30 years, mouse models have been produced in which the expression of genes encoding steroid hormone receptors has been enhanced, partially compromised or eliminated. These mouse models have revealed many of the physiological processes regulated by androgens, estrogens and to a more limited extent glucocorticoids in the testis and male accessory organs. In this review, advances provided by mouse models that have facilitated a better understanding of the molecular regulation of testis and reproductive tract processes by steroid hormones are discussed. Androgen is essential for the development of testes and the male reproductive tract, as well as maintenance of male fertility. Testosterone produced by Leydig cells is the major androgen responsible for androgenic effects in the testis and the male reproductive tract that has access to testosterone in fluid derived from the testis. In male accessory organs not exposed directly to testicular fluid, conversion of plasma-derived testosterone to dihydrotestosterone (DHT), the reduced and more potent form of testosterone, contributes to androgen actions. Androgens are ligands for androgen receptor , a 110 kDa intracellular protein encoded by a single gene on the X chromosome. Upon androgen binding, AR is released from sequestering heat shock proteins in the cytoplasm and either translocates to the nucleus to regulate gene expression or to the plasma membrane to activate kinase signaling pathways. In the absence of androgen or AR, male accessory organs do not develop. In the testis, lack of testosterone or AR causes spermatogenic arrest during meiosis, resulting in infertility. Global and cell-specific alteration of AR expression has provided important information revealing the mechanisms by which androgen and AR support organ development and function [Ar gene is present on the X chromosome, mice with a naturally occurring dominant testicular feminization mutation (Tfm) in the Ar gene provided a male global AR knock out model that was studied for 30 years before inducible gene elimination technology was developed [Cre) driven by the β-actin or phosphoglycerate kinase (PGK) promoter resulted in global AR knockout (ARKO) mice lacking AR in all cells [Tfm male mice have an external female-like appearance with anogenital distance similar to females, indicating a lack of androgen activity to provide for masculinization during fetal development. Structures derived from the Wolffian ducts  or urogenital sinus  are absent. Fallopian tubes and uterus are not observed, and no vaginal opening is found in ARKO males, but Tfm male mice have a short blind-ending vagina, similar to what is seen in humans with natural mutations of AR. Testes are 20% of wild-type size and not descended into the scrotum. No haploid germ cells are present and there are few meiotic germ cells. These mouse models confirmed earlier androgen ablation studies showing that testosterone signaling was required for spermatogenesis and male fertility, but cell-specific mechanisms were not identified . A summAr exon 2 was eliminated using a tamoxifen-inducible Cre recombinase [A different strategy to investigate AR functions in adults was employed to develop the iARKO mouse, in which ombinase . After 5The use of Cre transgenes driven by cell-specific promoters to produce conditional knockout mice allowed identification of AR actions in specific cells and tissues. Initial studies employing selective knock out of AR in Sertoli cells using Cre recombinase expressed from the Sertoli cell specific anti-Mullerian hormone (AMH) promoter allowed normal development of urogenital tracts, and normal testis descent into the scrotum. Germ cell entry into meiosis was normal. However, spermatogenesis was arrested during the pachytene or diplotene stages of meiosis prior to the first meiotic division. Additionally, fewer seminiferous tubules displayed lumens, indicating that fluid secretion by Sertoli cells was impaired ,8. TestiFollow-up studies of SCARKO mice revealed additional structures and processes in Sertoli cells needed to maintain fertility require androgen signaling. Formation of the blood–testis barrier (BTB), including correct temporal expression and localization of tight junction proteins to regions of cell–cell adhesion, was delayed and decreased in SCARKO mice. Eventually, the BTB formed in about 85% of tubules of adult SCARKO mice , but regThe critical necessity for AR activity in Sertoli cells was confirmed using comparisons of hypogonadal (hpg) mice lacking circulating gonadotropins and having low androgen levels versus hpg.ARKO or hpg.SCARKO mice having the additional absence of AR in all cell types or specifically in Sertoli cells, respectively. These models were used to show that androgen stimulation of spermatogonia and spermatocyte development requires direct action on Sertoli cells because androgen induced seminiferous tubule and germ cell development only in hpg mice .Ar null mutant in Sertoli cells that resulted from an inversion of exon 1. The mouse had a phenotype similar to SCARKO mice lacking exon 2 or 3. A hypomorph mouse line was also produced in which AR activity was reduced in Sertoli cells due to the insertion of the neomycin gene into exon 1 of the Ar gene. Expression of the Rhox5 testosterone activity marker was reduced by 80% in the Ar null model but a 45% reduction in expression of the testosterone activity marker was observed in the hypomorph suggesting that partial AR activity was retained. In contrast to models lacking Ar exons, the hypomorph model was able to maintain meiosis  suggesting that meiosis can be completed despite reduced AR-mediated signaling. Production of spermatids in the hypomorph model allowed studies of testosterone signaling on spermatid development. These results confirmed earlier hormone deprivation studies showing that AR function in Sertoli cells is required for post-meiotic functions including adhesion of spermatids to Sertoli cells during their initial stages of elongation and release of mature elongated spermatids from seminiferous epithelium [The Braun lab produced an ithelium .Abpa) gene promoter. In these mice, AR expression was much stronger than normal in Sertoli cells two to five days after birth when AR production is usually first detected. TgSCAR mice had testes up to 40% smaller than normal due to a reduction in the numbers of post-natal Sertoli cells at each developmental stage from post-natal day (PND) 5 to 70. Tubule lumen formation (a measure of Sertoli cell maturation) occurred earlier and expression of AR-mediated gene expression was greater in TgSCAR testes. The ratio of spermatocytes and spermatids to Sertoli cells was also higher at earlier developmental timepoints (PND 10-30) indicating accelerated spermatogenic development. These results support the idea that precocious AR expression causes earlier Sertoli cell maturation in the TgSCAR mouse [Effects of precocious AR expression in Sertoli cells were investigated in the transgenic Sertoli cell androgen receptor (TgSCAR) model mouse in which an AR transgene was expressed specifically in Sertoli cells using the androgen-binding protein  was not altered whereas, other mRNAs  were severely reduced. These results indicate that the SPARKI model can be used to differentiate members of the two classes of AR regulated genes.Information about AR interactions with androgen response elements (AREs), DNA sequences regulating gene expression, was provided by the SPARKI mouse model . This moLhr mRNA, which might account for the 25% reduction of testicular androgens. These results suggest that signals downstream of GR in Sertoli cells partially enhance Leydig cell function and reinforce the idea that Sertoli cells communicate with and maximize effectiveness of Leydig cells. Because GR can be detected in post-natal but not adult Sertoli cells, the SCGRKO phenotype may represent developmental defects that lead to lasting effects [The SPARKI mouse model results suggested that glucocorticoids acting via GR can contribute to regulation of some AR-mediated gene expression in Sertoli cells. Consistent with this idea, the Sertoli cell GR knock out (SCGRKO) mouse model provided evidence that signals downstream of GR may modulate functions supporting spermatogenesis. Weights of testes, epididymis and seminal vesicles were normal and SCGRKO males were fertile with normal litter sizes. However, the percentage of patent seminiferous tubules was reduced, and tubular diameters were inexplicably increased in SCGRKO mice. Numbers of Sertoli cells, spermatocytes and round spermatids were similarly reduced (25–30%) with Sertoli-germ cell ratios remaining normal. Leydig cell numbers were normal but there was Leydig cell hypertrophy, accumulation of lipid droplets in some Leydig cells, altered expression of some Leydig cell transcripts required for steroidogenesis and a reduction of  effects .Early studies of AR-regulated genes identified using SCARKO mice were reviewed previously ,31. ThesAr mutant testes that differed by more than 2-fold, with about twice as many being downregulated by AR as upregulated. Genes involved in metabolic processes and signal transduction were identified as major responders to the loss of AR activity. About 65% of the AR regulated genes were associated with an ARE within 6 kb of their transcription start sites and 23% were previously shown to be regulated by AREs suggesting that many AR-regulated genes are directly regulated by AR binding to AREs [A follow-up study of the AR hypomorph models using mouse genome arrays detected 62 transcripts in  to AREs .Rpl22 transgene encoding a ribosomal protein that is tagged with a hemagglutinin (HA) epitope in response to expression of Cre recombinase. This strategy allowed isolation of ribosome-associated (actively translated) mRNAs in specific cell types in the presence or absence of AR expression. Using Leydig cell-specific (Cyp17iCre) or Sertoli cell-specific (AMH-Cre) transgenes with intact endogenous AR expression and a microarray approach for mRNA analysis, numerous LH-regulated, ribosome-associated mRNAs were identified in Leydig cells. However, few differentially regulated translated mRNAs were found to be enriched in Sertoli cells of adult mice after a four-hour stimulation with testosterone [Ribotag mice harbor an osterone .Using RiboTag mice having AR knocked out in Sertoli cells and an RNA-seq assay allowed the identification of a large repertoire of new and previously undetected AR-regulated translated mRNAs in PND 10 Sertoli cells and indirectly regulated mRNAs in other cells. Approximately 1000 mRNAs were found to be differentially expressed after elimination of Sertoli AR, with slightly more down-regulated than up-regulated in total testis. However, Sertoli cell-specific mRNAs were more often down-regulated by testosterone via AR because 60% of Sertoli mRNAs regulated 2-fold or more are up-regulated in the absence of Sertoli AR . This fiEmployment of scRNA-seq technology with SCARKO mice added to information provided by the RiboTag mouse models about Sertoli cell- and germ cell-specific genes and pathways that are altered downstream of testosterone and AR signaling. Using scRNA-Seq and 20-day-old SCARKO mice, 347 up-regulated and 250 down-regulated genes expressed in Sertoli cells were identified, again indicating that most DEGs were downregulated by testosterone and AR signaling. However, it was not yet known whether AR directly down-regulates a larger population of genes or whether many testicular genes are down-regulated downstream of initial AR-mediated gene expression changes in Sertoli cells. At least 100 of the identified DEGs are known be associated with spermatogenesis, apoptosis, meiotic cell cycle, reproductive processes, male gamete generation germ cell development and cell migration. Transcriptome analysis showed that expression of cell migration/growth genes is a major alteration occurring in the absence of AR that may be an important function required for productive Sertoli cell signaling. Pseudotime trajectory analysis indicated that Sertoli cell development and maturation was affected by the loss of AR. The scRNA-seq strategy determined that lack of AR in Sertoli cells did not decrease the number of differentiated spermatogonia developing from germ line stem cells but, expression of over 100 spermatogonia mRNAs were altered including those associated with germ cell development and spermatogenesis .The Handel group used scRNA-seq and SCARKO mice in which all licensed germ cells were synchronized to enter meiosis concurrently to examine the regulation of meiotic progression by Sertoli cell AR . These sCombining a scRNA-seq strategy with germ cell synchronization in SCARKO testes, it was found that differential gene regulation in germ cells begins in differentiated spermatogonia and leptotene/zygotene spermatocytes. Transcriptomic arrest occurs in most SCARKO germ cells during leptotene/zygotene. However, cell development continues to a mid-pachytene state, implying that cytological progress is uncoupled from the molecular gene regulatory environment. These results indicate that androgen signaling from Sertoli cells is critical throughout early meiotic prophase and for transition to the divisional phase of meiosis. Down-regulated genes identified in SCARKO testes included those associated with cytoskeleton, acrosome or cilia organization and formation that are required for post-meiotic germ cell development. This finding raises the question of whether the lack of post-meiotic spermiogenesis gene expression is a contributing signal to meiotic arrest or simply results from germ cells that are not destined for further development. Upregulated SCARKO mRNAs included those for mitochondrial membrane and oxidative phosphorylation, which may be due to germ cell disruption or decreased ability to repress oxidative phosphorylation in SCARKO testes. A relatively small number (165) of Sertoli cell mRNAs were differentially regulated by more than 2-fold. Of the 165 DEGs, 77 were previously reported in the RiboTag/SCARKO mice study  that didRad51 recombinase to DNA breaks. Absence of recombination and repair and the incomplete synapsis of chromosomes are known to activate cell checkpoint responses in meiotic germ cells that act later to halt meiosis during the pachytene stage prior to the first meiotic division [The Liu lab used SCARKO mice to study processes in meiotic germ cells that require androgen-mediated signals from Sertoli cells . In SCARdivision ,37,38,39SCARKO testes were also found to express elevated levels of EGF family growth factors, suggesting that signaling downstream of AR normally limits production of EGF family members. The finding that an inhibitor of EGF receptor activity partially rescued meiotic progression in EGF-overexpressing mice provided more support for the idea that androgen-mediated limitation of EGF production during specific stages of germ cell development may be required to maintain meiosis 2+ concentrations and the PI3K/AKT and MAPK/ERK protein kinases, as well as gene expression [Steroid hormones act by at least two intracellular mechanisms. In the classical or genomic pathway, steroid hormones bind their specific receptors, which dimerize to form a DNA binding complex that regulates gene expression. In the nonclassical or nongenomic pathway, steroid hormones act via receptors associated with the internal side of the plasma membrane to modulate kinase pathways to directly alter cellular processes, such as cAMP concentrations, Capression .Ar gene, were expressed in a correct temporal and cell-specific manner and did not disrupt spermatogenesis or fertility in the presence of endogenous AR [Understanding relative functions of classical vs. nonclassical testosterone signaling in Sertoli cells was impeded by a lack of experimental systems to identify testosterone actions mediated by each pathway. To study contributions of each signaling pathway in vivo, unique transgenic mouse models were produced that expressed a wild-type AR transgene or transgenes having independent mutations incorporated into the AR transgene . In the enous AR ,40.To study functions of individual testosterone pathways in Sertoli cells, endogenous AR was knocked out specifically in these somatic cells on gestation day 16.5 (GD 16.5) using AMHCre. Testis weights of mice lacking AR in Sertoli cells but having both AR-C and AR-NC or AR-C alone were 87% and 81% of wild type, respectively. However, AR-NC expression alone did not alter testis weight (26% of wild-type) above that of SCARKO mice. The gross appearance of other reproductive tract tissues including seminal vesicles  were unaffected by the Sertoli cell-specific changes in AR activity. Spermatogenesis was not disrupted in mice expressing both AR-C and AR-NC. In AR-C mice, spermatogenesis could be completed. However, 50% of AR-C seminiferous tubules showed evidence of inefficient spermatogenesis, including tubules with no or prematurely released germ cells, tubules lacking lumens, disorganized germ cell positioning and mis-localization of elongated spermatids. These findings suggest that AR-C requires additional AR-NC activity for fully efficient spermatogenesis and support of germ cell development, differentiation and motility including the proper migration of elongated spermatids. Spermatogenesis could not be completed but was maintained up to the round spermatid developmental stage in 36% of AR-NC mice, indicating that AR-NC activity in Sertoli cells contributes to the completion of meiosis and that AR-C is required for elongated spermatid development. Together, the pathway-specific AR transgene studies show that AR-C and AR-NC testosterone pathways synergize in Sertoli cells to permit fully efficient spermatogenesis and fertility .Syce1, Syce2 and Syce3) encoding essential components of the synaptonemal complex central element and 2 members of the cohesion complex  needed for synaptonemal complex formation and completion of meiosis. In agreement with Larose and colleagues [A survey of germ cell mRNAs essential for completion of meiosis in SCARKO and SCARKO + AR-C testes revealed that AR-C was required for expression of three mRNAs  the two signaling pathways had complementary functions that together provided optimal male fertility, (2) nonclassical signaling contributes to BTB maintenance, spermatid adhesion plus migration and localization of elongated spermatids and (3) classical signaling contributes to BTB maintenance, transition of round to elongated spermatids and elongated spermatid development and is required to complete meiosis including inducing mRNAs required for chromosome synapsis .In mice with global knockout of endogenous AR, restoration of AR-C signaling alone provided 76% of normal androgen activity during fetal development as measured by relative anogenital distance, whereas AR-C plus AR-NC together provided for full androgen-dependent development. Testosterone signaling through AR-C plus AR-NC or AR-C alone, but not AR-NC alone, were sufficient for production of mature elongated spermatids/sperm. However, none of the mice lacking endogenous AR were fertile because efferent ducts, epididymis and ductus deferens did not develop and thus sperm delivery was not possible. This result suggests that development of these accessory reproductive organs is more sensitive to testosterone signaling than is the testis. Testosterone-dependent prostate lobe development was partially supported by AR-C, but AR-C plus AR-NC synergism supported 2-fold additional development of prostate lobes to 57% to 74% of normal . These dSertoli cells are not the only critical somatic cell type supporting spermatogenesis, as shown by results from the PTM-ARKO mouse model that eliminated AR from smooth muscle cells including PTM cells surrounding the seminiferous tubules and in blood vessels. Although Cre recombinase expression driven by the smooth muscle myosin heavy chain (Myh6) promoter could be detected in only about 40% of PTM cells, male PTM-ARKO mice did not sire any pups, became azoospermic and progressive loss of spermatogonia and spermatocytes was more dramatic than observed in adult SCARKO mice. Sertoli cell numbers did not vary between PTM-ARKO and SCARKO mice but expression of AR-dependent mRNAs in Sertoli cells was decreased and Sertoli cell nuclei orientation in PTM-ARKO testes became progressively more disorganized, with Sertoli cells often mis-localized in the middle of the seminiferous tubules by PND 100. One possible mechanism responsible for disruption of Sertoli cell polarization and attachment to the basement membrane in PTM-ARKO mice may be due to disrupted deposition of extracellular matrix by PTM cells, that in turn alters Sertoli cell function .−/y, with AR excised in PTM cells used the Cre transgene driven by the transgelin (Tagln) smooth muscle protein 22-a promoter. This model had normal fertility although testes weight was reduced 34%, total germ cell numbers decreased and epididymal sperm numbers were reduced 57%. Expression of Sertoli cell marker genes including those required for cell–cell interactions and genes required for PTM cell contraction was decreased. Reduced sperm production in PM-AR−/y and PTM-ARKO mice may partially reflect altered Sertoli cell gene expression [−/y fertility phenotype may be due to lower efficiency of the smooth muscle protein 22-a driven Cre transgene in the testis [A second mouse model, PM-ARpression . The lese testis . The strIn PTM-ARKO mice, Leydig cell numbers and AR expression were not altered but intratesticular (but not serum) testosterone levels were increased. LH levels were increased, perhaps as a compensatory mechanism to stimulate testosterone production from Leydig cells . Further−/Y) mice showed that functional AR in Leydig cells is essential to maintain spermatogenesis, testosterone production and fertility. Using an AMH receptor-Cre (AMHR-Cre) strategy to eliminate AR in Leydig cells and partially in Sertoli cells, two patterns of seminiferous tubules were observed. One showed a maturation arrest phenotype with significant deficiency of AR in both Leydig and Sertoli cells. A second Sertoli cell-only phenotype lacking germ cells was also observed in which most Leydig cells were negative and Sertoli cells showed relatively weak AR immunoreactivity [−/Y model and the loss of AR in some Sertoli cells limits interpretation of the results.Leydig cell AR knock out  of ARKO Leydig cells did not increase ,44. AlthDhh-Ptch1, Pdgfa-Pdgfra, Amh-AMhr2) are induced in TgSCAR mouse testes. Intratesticular testosterone levels per Leydig cell were elevated in TgSCAR mice [A scRNA-Seq study of SCARKO mice also determined that numbers of mature Leydig cells were reduced, whereas interstitial progenitor and immature Leydig cells accumulated . This stCAR mice . ResultsComparisons of hpg, hpg.ARKO and hpg.SCARKO mouse models showed that signals from testicular ARs during fetal development inhibit an inappropriate LH-dependent developmental pathway in Leydig/adrenal cell precursors that results in cells with adrenal characteristics. This result may provide one explanation for Sertoli cell AR-mediated support of the Leydig cell population. The hpg-based models also showed that LH acts to induce the steroidogenic potential of Leydig cell precursors but additional stimulation via ARs is essential for induction of the Leydig cell phenotype .−/y mouse model did not alter sperm count, fertility, seminiferous tubule structure or the relative numbers of germ cells at various stages of development. This mouse model provides the important finding that meiotic and post-meiotic germ cell AR, if expressed, is not required for spermatogenesis or male fertility [Excision of AR during early meiosis in leptotene-zygotene germ cells using the Sycp1-Cre transgene in the G-ARertility .Together, results from the knockout mouse models revealed that there is an interdependence between testicular cells that is required for cell maturation and maintenance of spermatogenesis. Testosterone-mediated signals from Sertoli and PTM cells contribute to proliferation and maturation of adult Leydig cells while facilitating steroidogenesis. PTM cells provide extracellular matrix and other cell–cell contact signals to improve Sertoli gene expression and polarization. Finally, AR-mediated signals from Sertoli cells support maintenance of the blood–testis barrier, meiosis, spermatid attachment and sperm release .Arr2pb) including two androgen response elements (AREs) from the promoter to drive Cre expression and selectively eliminate AR expression in prostate epithelium as well as epididymis, seminal vesicles and ductus deferens. Loss of AR in prostate epithelium was progressive, becoming nearly total by 24 weeks postnatal. In the prostate epithelium ARKO (PEARKO) mouse model, the macroscopic prostate structure appeared normal but prostate lobe weights were uniformly reduced [Functions of AR in epithelial cells of prostate were examined using two similar mouse models. These models employed a composite probasin promoter -Cre. In these mice, ventral prostates were smaller, epithelial cells were more cuboidal and flattened, epithelial proliferation was decreased and apoptosis increased in older mice. There was also dramatically decreased collagen deposition on the FSP1-ARKO prostate basement membrane, which may contribute to defects of epithelial development and growth. FSP1-ARKO mice also had down-regulated expression of FGF-10, FGF-7 and IGF-1 that act as prostate stromal growth factors in the prostate . TogetheA double stromal AR knock out (dARKO) mouse model was created by mating SM-Cre with FSP1-Cre mice. Offspring lacking AR in prostate smooth muscle and fibroblasts had smaller anterior prostate lobes, but ventral and dorsal lateral lobes were similar to controls. Branching structure was reduced in anterior and dorsolateral but not ventral lobes. Continuing loss of infolding was initiated by PND 56. Together, these results suggest that stromal/mesenchymal AR is essential for prenatal organogenesis and maintaining homeostatic balance of prostate in postnatal and adult stages. Proliferation of epithelial cells in 4- and 8-week-old dARKO prostates was decreased, but in 11- and 24-week-old mice the very low proliferation rates were similar to controls. Apoptosis rates in prostates through 16 weeks after birth were low and normal but, were increased above normal in 24-week-old dARKO mice. dARKO mice also had decreased height of the luminal epithelium with flattened and cuboidal epithelial cells. Thus, epithelial cell proliferation and proliferation is dependent upon stromal AR-derived signals in young mice and this signaling is required to maintain epithelial survival in adults. Cell lines derived from dARKO prostate were used to identify IGF-1, PGF and SPP1 as AR-regulated stromal paracrine growth factors .In PEARKO mice, AR activity was also reduced in epithelium of epididymis and vas deferens and unexpectedly in smooth muscle cells of seminal vesicles due to induced expression of Cre recombinase by the probasin promoter in these organs. Seminal vesicle weights were reduced by 55% from decreased secretion and reduced weight of the emptied gland. Epithelial cells in the proximal region were less folded, lower and cuboidal, and contained little cytoplasm. The smooth muscle layer was reduced about 50% and disorganized. These results indicate that testosterone-mediated signals from smooth muscle cells are important to maintain normal epithelial and stromal cell function including secretion. Androgen signaling in smooth muscle may limit epithelial cell proliferation in seminal vesicles, because the number of epithelial cells undergoing mitosis in mice lacking AR in seminal vesicle smooth muscle cells was increased 3-fold, resulting in hyperplasia.In other studies employing PEARKO mice, the numbers of copulatory plugs, which are dependent upon secreted proteins from seminal vesicles, were similar to normal 4 h after coitus but were smaller with a soft and fibrous consistency that likely is responsible for the 70% decrease in plug-positive females when assessed the morning after coitus. Fertility of PEARKO males was reduced, with litter size and number reduced. The success of in vivo fertilization was reduced almost 3-fold and in vitro fertilization was reduced at low sperm concentrations, perhaps due to a mild defect in sperm binding to the zona pellucida. Sperm numbers were unchanged, but sperm released from PEARKO cauda epididymis had increased percentages of abnormal bent tails with hairpin structures and displayed hyperactive, erratic, and whiplash-like movement of capitated sperm . DisruptThe PTM-ARKO model also provided information about AR functions in seminal vesicles. The reproductive tract formed normally. However, in the absence of smooth muscle AR, seminal vesicle weight was reduced after PND 21. The epithelium was cuboidal with an increase in branching, and there was reduced smooth muscle thickness. These results indicate that testosterone-derived signals from smooth muscle cells are important to maintain normal seminal vesicle epithelial and stromal cell function including secretion. As in PEARKO mice, epithelial cells undergoing mitosis in PTM-ARKO were increased 3-fold, resulting in hyperplasia. Further studies revealed that 17β-estradiol (E2) caused more pronounced hyperplasia of the epithelium and increased smooth muscle thickness in the PTM-ARKO. The results indicate that in the absence of testosterone, estrogens can cause uncontrolled proliferation and hyperplasia in seminal vesicles .In the PEARKO model, epididymal morphology was normal, but epididymal weight was reduced about 20% and stroma thickening was present in the corpus and cauda regions. Due to reduced androgen action in the epididymis, sperm may transit more rapidly through the caput and corpus regions, leading to sperm maturation defects and increased rates of spontaneous acrosome reaction in PEARKO sperm ,48.Rnase10 gene promoter (ProxE-ARKO) resulted in regression of the epididymal initial segment, epithelial hypoplasia and hypotrophy and epididymal obstruction beginning at PND 20-25. Blockage of fluid flow through the epididymis caused back pressure into the efferent ducts and testis resulting in atrophy of the seminiferous epithelium, orchitis, fibrosis of testicular parenchyma and azoospermia [Two mouse models were used to study AR function in the proximal region the epididymis. Knock out of floxed AR in principal cells of the proximal (caput) epididymis using Cre recombinase driven by the ospermia .Rnase10 Cre. Together, the two epididymis AR knock out models confirm the requirement for AR and androgen signaling in proximal epithelial cells to allow development and maintenance of the initial segment of epididymis as well as fluid and sperm flow from the testis through the epididymis to maintain fertility.A second mouse model, the caput epithelium androgen receptor knock-out (CEARKO), employed a FoxG1 Cre to knock out AR in principal cells of the proximal epididymal epithelium . In CEARAndrogens regulate the differentiation, development and adult function of the male reproductive tract, as described in detail in the initial part of this article. These findings have resulted in all facets of androgen production and action being a major focus of research in the area of male reproductive biology. Despite the primacy of androgen in terms of sex steroid regulation of male reproduction, a literature dating back almost a century has also clearly shown that estrogens are critical for male reproduction.Evidence for the existence of an ovarian hormone that we now know was an estrogen was obtained in the early 1920s, and by 1940 E2 and other estrogens had been identified ,50. InitOnly a year after the initial report of estrogens in the male, Burrows  reportedThe discovery that developing males were even more sensitive than adults to estrogens led to the development of an extensive literature showing long-term deleterious effects of fetal or neonatal estrogen exposure on the testis and many other male reproductive organs . The litA separate literature on circulating estrogen concentrations in males and the expression of estrogen receptors in the reproductive tract and elsewhere in the male that arose in parallel with the studies of exogenous estrogen effects in males provided additional data supporting an estrogen role in normal male reproduction. However, this remained speculative for years, as a definitive experimental system that allowed this hypothesis to be tested was unavailable. Estrogen is readily measurable in the peripheral blood of males from many animal species  mouse by Lubahn, Korach and Smithies provided a powerful and novel tool that allowed the consequences of ESR1 loss in males to be determined [Esr1KO males, as well as other model systems, such as the aromatase knockout mouse that does not produce estrogen, additional actions of estrogen in the epididymis, as well as non-reproductive organs such as adipose tissue and the thymus, have been demonstrated [The development of the estrogen receptor 1 knockout  signal through the canonical pathway involving ligand binding to their cognate receptors, dimerization of liganded receptors and translocation of ligand-receptor complexes to DNA. These complexes bind to estrogen-responsive elements in DNA, altering gene transcription and inducing the ultimate hormonal effects on the cell. In addition to nESR1, a small fraction of ESR1 protein produced (approximately 5–10%) is palmitoylated and routed to the cell membrane. This membrane ESR1 (mESR1) signals through tyrosine kinase and other pathways totally distinct from nESR1 (reviewed in ). For maEsr1KO mice [Esr1KO, as one might expect; in contrast to the infertility of Esr1KO mice, NOER mice sometimes sired one litter as they reached puberty but then became infertile. These results indicate that mESR1 plays critical roles in the male reproductive tract, and that loss of membrane ESR1 signaling alone results in infertility.In 2014, two groups developed mouse models that maintained normal nESR1 signaling but had minimal or absent mESR1 signaling ,28. The 1KO mice . The pheRecent years have seen rapid progress in our understanding of the role of endogenous E2/ESR1 signaling in males and resulted in identification of critical estrogen actions in normal development and function of reproductive and non-reproductive organs in males. Relatively new work on the actions of 17α-E2, a stereoisomer of the widely studied 17β-E2, suggests that this estrogen, which previously received scant attention from the research community, can have important and unprecedented effects on male longevity. This provocative and unexpected line of work clearly emphasizes the pleiotropic effects of estrogens and demonstrates that our understanding of estrogens and their effects in the male is still in its infancy.In 2014, Harrison et al.  reportedDespite intensive research, the reason for the sexually dimorphic nature of the 17α-E2 effect, where longevity increases only in males, remains unclear but is believed to result from the high circulating 17β-E2 concentrations in females, which may mask the 17α-E2 effects that can be detected after treatment of males. Another obvious question posed by the longevity increases in males treated with 17α-E2 was the mechanism of these effects. 17α-E2 has not been studied intensively, but data from several laboratories indicate that 17α-E2 has much less affinity for nuclear ESR1 than does 17β-E2 . In addiEsr1KO mice [If 17α-E2 has minimal effects through ESR1, maybe it induces its longevity effects through another receptor. It was suggested in the early 2000s that 17α-E2 could act through another putative estrogen receptor, designated ER-X, to produce its effects ,68. Howe1KO mice . These dWork to date has not noted any obvious effects on reproductive tract structure or function in the males treated with 17α-E2, but this has not been examined in detail. It is also not clear if there is an endogenous role of 17α-E2 in the male or female, even though available information suggests that significant amounts of 17α-E2 may be present in men and women. Clearly, we need much more information to fully understand the role of 17α-E2 in the male, as well as the female.Esr1KO and NOER mouse began to provide tools to definitively evaluate the roles of ESR1 and the membrane component of its signaling and provide definitive answers to questions that could not be effectively answered earlier. The recent results with 17α-E2 emphasize that there is much that we do not know about the roles of estrogen in the male, despite rapid progress in this area over the past few decades. Future work in this area will likely reveal important additional insights into what estrogens do in males, and our understanding of the role of estrogens in the male is likely to keep evolving in the coming years.Three decades ago, the question of whether or not there was an important role for endogenous estrogen in the male was still being debated, although the ideas that males made estrogens, that exogenous estrogens could produce effects in males, and that males had receptors for estrogen were well established. We now know that not only is signaling through ESR1 necessary for the normal development and function of the male reproductive tract and fertility itself, but the loss of even the membrane form of ESR1 in males is sufficient to render these animals infertile. This finding is something that would have seemed inconceivable in the years before the development of the Testosterone has been known to be essential for male fertility for nearly a century. Later testosterone ablation studies demonstrated that the steroid hormone was required for the development of the testis and accessory sex organs as well as completion of spermatogenesis. However, transgenic mouse models focusing on the activity of AR, GR or ERs in specific testicular cells have allowed molecular characterization of steroid hormone actions including the identification of specific targets of steroid hormone action that are required to maintain fertility. Without knockout mouse models, the non-intuitive discovery that estrogen signaling was required for fluid resorption in the efferent ductules would not have been possible. Studies employing mouse models also have identified intercellular signals that provide for collaborative support between testis cells to optimize spermatogenesis. In addition, use of mouse models allowed identification of signaling pathways and gene clusters that are regulated by steroid hormones in the testis. Together, these findings have characterized the matrix of signals required to maintain male fertility, allowing for the development of diagnostic tools to identify the causes of male infertility and treatments to correct errors that result in infertility.


Areal density of putative AR structures was higher in LN metastases than in primary tumors. Variation of the areal density within different LN metastases of a single patient was lower than between metastases of different patients. Abundance of putative AR structures in LN metastases was associated with shorter cancer specific survival (p = 0.013), and this association was independent of T and N stages. Abundance of putative AR structures in primary tumors and LN metastases had a cumulative adverse effect on prognosis. Enrichment of putative AR subpopulations in LN metastases suggest that in metastasis formation, there is a selection favoring cells capable of forming these structures. Higher intra-case constancy relative to inter-case variation suggests that such selection is stable in metastasis development. Our findings indirectly support the biological validity of our concept of putative AR structures.Anoikis refers to apoptosis induced by the loss of contact with the extracellular matrix. Anoikis resistance is essential for metastasis. We have recently shown that it is possible to quantitatively evaluate putative anoikis resistant (AR) subpopulations in colorectal carcinoma (CRC). Abundance of these multi-cell structures is an independent marker of adverse prognosis. Here, we have quantified putative AR subpopulations in lymph node (LN) metastases of CRC and evaluated their prognostic value and relationship with the characteristics of primary tumors. A case series included 137 unselected CRC patients, 54 with LN metastases. Areal densities (structures/mm Colorectal carcinoma (CRC) causes 1 in 10 of global cancer deaths . The 5-yAnoikis is a subtype of programmed cell death, where an epithelial cell dies after detachment from the extracellular matrix (ECM) . Physiol. However, it has not been possible to evaluate anoikis resistance by using conventional tissue sections. We have recently shown that transfection of Caco-2 cells with mutant KRAS or BRAF genes modifies both the measured anoikis resistance and induce a characteristic change in the 3D cell culture growth pattern. We found that native Caco-2 cells show a low level of anoikis resistance and in 3D cultures form cysts with a single layer of columnar cells, all with a contact with the ECM. In contrast, the transfected Caco-2 cells show a high level of anoikis resistance, and in 3D cultures form partially filled cysts with the inner cells detached from the ECM but still maintaining resistance to anoikis , absence of association between the total AR counts and distant metastasis, a dominant prognostic factor, suggests that the effect of putative AR structures might be independent even in terms of distant metastasis. The prognostic effect of putative AR structure amount in LN metastases is a novel finding which should be confirmed with an independent case series. In addition, the current cut-off values for high and low areal density should be considered provisional, likely needing adjustment based on additional case series. However, areal density of putative AR structures could complement the list of metastasis-based prognostic features such as N category, extranodal extension and LN ratio , 34. IntFor advanced stage CRC, only a limited number of predictive biomarkers are available, and validated predictive markers for adjuvant chemotherapy in stage III are lacking , 35. If Although our concept of putative AR structures represents a biologically plausible explanation for the prognostic associations we observed, actual anoikis resistance can only be detected in in vitro experiments . TherefoExtranodal invasion in LN metastases is a rather novel prognostic factor in several carcinoma types including CRC . We obseOur study represents a comprehensive assessment of the 137 patients of which 57 had LN metastases and 45 were examined. Due to the limited number of metastatic cases, our observations should be confirmed with an independent case series. However our conclusions are supported by systematic analyses of all LN metastases. The assessment was blinded for both clinicopathological features and the results of putative AR structure analyses of the primary tumors. Quantification of areal density using WSIs with proper software tools enables a higher detail of measurement than conventional light microscope analysis. Also, the reproducibility of the quantification method was confirmed in our previous paper [Our comprehensive analysis of LN metastases in CRC shows that putative AR structures are enriched in LN metastases as compared with primary tumors. As this could be related with a survival benefit of malignant cells linked with true anoikis resistance, the finding lends some support to the concept of putative AR structures being related with true anoikis resistance. Abundance of putative AR structures in nodal metastases is a new marker of poor prognosis.


The novel concept of immersive 3D augmented reality (AR) surgical navigation has recently been introduced in the medical field. This method allows surgeons to directly focus on the surgical objective without having to look at a separate monitor. In the dental field, the recently developed AR-assisted dental implant navigation system (AR navigation), which uses innovative image technology to directly visualize and track a presurgical plan over an actual surgical site, has attracted great interest.This study is the first systematic review and meta-analysis study that aimed to assess the accuracy of dental implants placed by AR navigation and compare it with that of the widely used implant placement methods, including the freehand method (FH), template-based static guidance (TG), and conventional navigation (CN).d statistical power analysis was used to investigate the effect size estimate and CIs of standardized mean differences (SMDs) between data sets.Individual search strategies were used in PubMed (MEDLINE), Scopus, ScienceDirect, Cochrane Library, and Google Scholar to search for articles published until March 21, 2022. This study was performed in accordance with the PRISMA  guidelines and registered in the International Prospective Register of Systematic Reviews (PROSPERO) database. Peer-reviewed journal articles evaluating the positional deviations of dental implants placed using AR-assisted implant navigation systems were included. Cohen P<.001) and CN method . However, the accuracies of the AR navigation and TG methods were similar .Among the 425 articles retrieved, 15 articles were considered eligible for narrative review, 8 articles were considered for single-arm meta-analysis, and 4 were included in a 2-arm meta-analysis. The mean lateral, global, depth, and angular deviations of the dental implant placed using AR navigation were 0.90 (95% CI 0.78-1.02) mm, 1.18 (95% CI 0.95-1.41) mm, 0.78 (95% CI 0.48-1.08) mm, and 3.96° (95% CI 3.45°-4.48°), respectively. The accuracy of AR navigation was significantly higher than that of the FH method (SMD=−1.01; 95% CI −1.47 to −0.55; The positional deviations of AR-navigated implant placements were within the safety zone, suggesting clinically acceptable accuracy of the AR navigation method. Moreover, the accuracy of AR implant navigation was comparable with that of the highly recommended dental implant–guided surgery method, TG, and superior to that of the conventional FH and CN methods. This review highlights the possibility of using AR navigation as an effective and accurate immersive surgical guide for dental implant placement. The clinical success of dental implant placement requires proper preoperative implant planning along with an effective and accurate surgical process ,2. CompuStatic dental implant–guided surgery has been widely used in clinical practice, because it results in highly accurate implant placement . HoweverCurrent dynamic dental implant–guiding systems often consist of 2 main components: an optical tracking device that records either passive or active tracking arrays positioned on patients and surgical instruments and a navigation system that displays the positions of the drill on CBCT images ,18. The Recently, augmented reality (AR)—an innovative image technology that produces an immersive surgical context by merging digital data with the real environment—has been incorporated into a dynamic dental implant–guiding system to help operators directly visualize digital navigation information over the actual operating field of view to improve real-time surgical performance -23. AR tTherefore, we investigated the accuracy of innovative imaging technology in the form of an AR-assisted navigation system that can merge digital images in a real environment to help oral surgeons visualize virtually planned dental implant positions at actual surgical sites. The primary aim of this study was to evaluate the accuracy of the recently developed AR-assisted dental implant navigation system (AR navigation) in terms of positional deviations between the planned and placed implants. The secondary aim of this study was to compare the accuracy of AR navigation with that of other dental implant placement methods, including the FH method, template-based static guidance (TG) method, and conventional navigation (CN) method.This study was performed in accordance with the PRISMA  guidelines  and regiPopulation (P): Dental implants, implant pins, implant drilling channels placed on humans or animals or simulated clinical modelsIntervention (I): Implant surgery guided by AR navigation (AR navigation group)Comparison (C): Implant surgery guided by other methods such as the FH method (FH group), TG method (TG group), and CN method (CN group)Outcomes (O): Positional deviations of the placed implant or drilling channel described by the differences between the planned and actual positions of the placed implantsStudy (S): Randomized and nonrandomized, observational , and preclinical or in vitro studiesJournal of Clinical Periodontology, Periodontology 2000, Journal of Periodontology, Clinical Oral Implant Research, and Clinical Implant Dentistry and Related Research were manually searched. The search strategy for each database was established using Boolean logic  to combine search terms. The individual Boolean operators established for searching for each data source are listed in The following electronic databases were searched: PubMed, Scopus, Web of Science, Cochrane Library, and Google Scholar. In addition, the references cited in the full-text and relevant articles in several dental journals such as This systematic review considered all original studies published until March 21, 2022  that evaluated the accuracy of AR navigation without any language restrictions. Studies without metric measurements for reporting implant positions and those that were unrelated to dental implant placement were excluded. Similarly, systematic or narrative reviews without implant placement accuracy assessments, letters to the editor, author or editorial opinion articles, and epidemiological studies were excluded. Gray literature and unpublished data were not included in this study to avoid a possible research bias. Only studies that reported deviation values between planned and executed implant positions were included in the meta-analysis. The detailed inclusion and exclusion criteria are listed in For study selection, 2 reviewers (HNM and DHL) independently screened and selected eligible studies based on inclusion and exclusion criteria. The agreement between reviewers was calculated using Cohen κ coefficient. Any disagreement was resolved through a discussion between the reviewers or consultation with an expert (VVD). Relevant information was extracted from the eligible studies, which included study characteristics , participant characteristics , AR system characteristics , the outcome of interest , comparison groups , and major findings from each included study. In case of missing or ambiguous data, the corresponding authors were contacted for data clarification. Information was extracted independently by 2 reviewers and recorded on an electronic spreadsheet . After the initial data extraction, the final data table was re-evaluated by both reviewers, and inconsistencies were corrected either by discussion between the 2 reviewers or by consultation with a third reviewer (VVD).The risk of bias in the included studies was assessed using the Risk Of Bias In Non-randomized Studies of Interventions tool  based onAll statistical analyses were performed using R statistical software , and the significance level was set at .05. Weighted bars and traffic light plots were used to visualize the results of the risk of bias assessments, and funnel plots were generated to visually report publication bias.The positional deviations between the planned and placed implants were analyzed using 6 outcome variables: lateral coronal deviation (LCD), lateral apical deviation (LAD), global coronal deviation (GCD), global apical deviation (GAD), depth deviation, and angular deviation .To obtain a general view of the accuracy of implant placement performed using AR navigation, single-arm meta-analyses for continuous data that involved estimating the mean deviations for each deviation variable were used for data analysis. Weighted means with 95% CIs were assessed, and subgroup analyses were performed according to deviation outcome variables .To compare the accuracy of AR navigation  with other implant placement methods (control groups), 2-arm meta-analyses for continuous data were performed. The standardized mean difference (SMD) with a 95% CI between the experimental and reference data sets was analyzed to estimate the effect size :mean is the difference in mean values between the experimental and control groups and SD of the measurements. Subgroup analyses were performed to compare the AR navigation group with the FH, TG, and CN navigation groups.where d2I statistic [Meta-analyses were performed using either a random effects model or a fixed-effects model based on the heterogeneity among studies evaluated by the Higgins tatistic :Q is the chi-square statistic and df is the degree of freedom. A higher 2I value indicates stronger heterogeneity across studies. In cases of I2≥50%, the data were considered statistically heterogeneous, and a random-effects model was used for data analysis.where The search results are shown in the PRISMA flowchart . In summThe characteristics of the included studies are summarized in Regarding AR technologies, most AR-assisted implant navigation systems allow the appearance of virtually planned implants. Some systems provide additional guidance such as the surgical path or drilling axis of the drills and the alveolar nerve or mandibular canal. Some systems included a warning function to alert surgeons and aid adjustments to the surgical strategy. In particular, one study  adopted The risk of bias assessment is shown in df=−4.604; P<.001). Visual inspection of the contour-enhanced funnel plot was performed to assess potential publication bias. The asymmetry in the plots, which might be owing to the increased missing data present in the contours of the statistically significant region (gray shaded regions) than in the areas of nonstatistical significance (white region), suggests that the cause of the asymmetry might be because of factors other than publication bias such as variable study quality [df=−1.097; P=.28).The funnel plot  and the  quality . After a quality , 11 dataA total of 346 implant sites were evaluated . Detailed descriptive characteristics of the AR-guided implant placement systems used in the included studies are presented in P<.001) and CN groups , and similar accuracy to the TG group . Detailed positional deviation comparisons are shown in P<.001; I2=47%). For global deviation (mm), the implants placed in the AR navigation group showed smaller deviations than those in the control group . The AR navigation group exhibited significantly smaller deviations than the FH group  and the TG group  but showed larger deviations than the CN group . Regarding the depth deviations (mm), the AR navigation group exhibited smaller deviations than the CN group . Angular deviations (°) of the implants placed in the AR navigation group were also significantly smaller than those of the implants placed in the control groups . Specifically, the AR navigation group showed smaller deviations of placed implants than the CN group  and the FH group  but exhibited larger deviations than the TG group .Forest plots representing the SMD between the implant positional deviations in the AR navigation and control groups are shown in This systematic review aimed to evaluate the accuracy of AR navigation and compare it with that of FH, TG, and CN methods. The results of the meta-analysis indicated that the errors of implant placement using AR navigation were within the safety zone, which is recommended to be in the range of 1-2 mm horizontally and vertically -51 and uThe accuracy of TG implant surgery is superior to that of the FH method . In addiSimilar to CN systems, AR navigation involves the same phase of preoperative implant planning based on CBCT and intraoral data of patients and requires a navigation system for tracking the real-time position of the surgical instruments. However, it uses innovative imaging technology in the form of AR to overlay the digital presurgical plan over the actual surgical site. Generally, the setting of an AR-assisted dental implant navigation system consists of 3 components: image registration, image tracking, and AR image display devices.Marker-based and marker-free methods are the 2 main approaches used for image registration and tracking. For marker-based image matching, specific artificial markers that are attached to the reference template applied in the patient’s mouth and implant drilling instruments are used to merge the digital presurgical plan with the real operation site. In contrast, the marker-free approach uses a point cloud–based registration method and tooth shape tracking methods to merge digital images in a real environment. In this review, most reported AR navigation systems relied on marker-based image registration and image tracking techniques -22,39-46As for AR image display, see-through HMD devices are the main tools ,42,44-46This systematic review has some limitations, including the relatively small number of included studies owing to the limited research in this new area and some sources of bias. Another limitation is the risk of high heterogeneity among the included studies. The inconsistency in the included studies was owing to the uniformity of implant placement error measurements, as multiple variables  were considered. The variety in the selection of the control group  and the subjects of the experiment  also contributed to the high heterogeneity among the studies. Therefore, in this review, a random effects model was used to perform a meta-analysis and subgroup analyses were performed to reduce heterogeneity. Further studies that consider patient- and surgeon-related factors, such as user satisfaction, convenience, and comfort, should be conducted to further our understanding of the existing AR-assisted implant navigation systems.This systematic review supports the relevance of AR-assisted implant navigation methods. The meta-analysis showed that the accuracy of AR implant navigation was comparable with that of the highly recommended dental implant–guided surgery method, TG, and even superior to that of the conventional FH and CN methods. It should be noted that although AR implant navigation may be considered an effective immersive surgical guidance for dental implant placement, limited studies regarding the clinical application are available. Further studies that consider patient- and surgeon-related factors, such as user satisfaction, convenience, and comfort, should be conducted to further our understanding of the existing AR-assisted implant navigation systems.