AR mRNA expression was higher under nutrient-deficient conditions and lower under hypoxic conditions in vitro. However, there were no differences in AR protein expression. No differences in AR mRNA expression were observed between the tested tumor structures taken from patients. No differences in AR mRNA expression were observed between the men and women. However, AR protein expression in tumors resected from patients was higher in the enhancing tumor region and in the peritumoral area than in the tumor core. In women, higher AR expression was observed in the peritumoral area than in the tumor core. AR expression in GBM tumors did not differ significantly between men and women, which suggests that the higher incidence of GBM in men is not associated with AR expression. In the group consisting of men and women, AR expression varied between the regions of the tumor: AR expression was higher in the enhancing tumor region and in the peritumoral area than in the tumor core, showing a dependence on tumor conditions (hypoxia and insufficient nutrient supply).Glioblastoma multiforme (GBM) is a malignant glioma, difficult to detect and with the lowest survival rates among gliomas. Its greater incidence among men and its higher survival rate among premenopausal women suggest that it may be associated with the levels of androgens. As androgens stimulate the androgen receptor (AR), which acts as a transcription factor, the aim of this study was the investigate the role of AR in the progression of GBM. The study was conducted on tissues collected from three regions of GBM tumors . In addition, an in vitro experiment was conducted on U-87 cells under various culture conditions , mimicking the conditions in a tumor. In both of the models, androgen receptor expression was determined at the gene and protein levels, and the results were confirmed by confocal microscopy and immunohistochemistry.  Glioblastoma multiforme is one of the most common gliomas ,2,3, accGBM in the early stages of growth is difficult to recognize and diagnose ,7,8. GBMThe gene-encoding AR is located on the long arm of the X chromosome , contains more than 90 kilobase pairs, and has eight transcripts. AR is nuclear receptor NR3C4  . AR protDespite intensive research, little is known so far about the regulation of gene expression by AR in GBM. In prostate cancer cells, the effect of AR on the biology of this tumor is very well understood . AndrogeThere are currently many studies analyzing the influence of genetic, epigenetic, and protein factors on the development and growth of GBM. Glioblastoma is a tumor whose characteristic mode of growth may exert an influence on the formation of different cell lineages in a single tumor. This is because it forms kinds of long protrusions when infiltrating healthy tissues . LimitedTo better understand the mechanisms leading to the development of GBM, it seems reasonable to conduct studies on isolated individual structures in the tumor. To better understand the impact of conditions in individual tumor structures and dynamic adaptive changes in the tumor, the present study introduced a cellular model using immortalized tumor-transformed glial cells. AR expression was analyzed in individual tumor structures resected from GBM patients as well as on cells in an in vitro model mimicking the conditions prevailing in the tumor, i.e., hypoxia, nutrient deficiency, and necrosis.The study group consisted of 24 people. Patients participating in the project were characterized by taking into account anthropometric factors such as gender, age, height, and BMI. Information was also recorded on environmental factors that affected individual patients, such as the type of job, smoking, and place of residence. The characteristics of the study group are shown in After the cells were cultured as stated, photographs of each culture were taken using a microscope attachment that protected the camera from varying positions and distances .In the presented set of photos, one can see the altered appearance of the cultures under each condition. Characteristic changes in the appearance of the cultures grown under the test conditions appeared at all repetitions of the experiment. Cells cultured under hypoxia formed clusters of cells; in addition, intercellular connections became less visible in all test conditions. VEGF-vascular endothelial growth factor) and apoptosis (CASP3-caspase 3) were performed  and CASP3 growth trends   .AR mRNA expression (relative to control) was statistically significantly higher in cells cultured under nutrient-deficient conditions (p = 0.00168) and statistically significantly lower under hypoxic conditions (p = 0.000000195) (Expressions were examined in U87 line cells cultured under different conditions . AR mRNA0000195) A. In con0000195) A.AR protein expression was determined using ELISA(enzyme-linked immunosorbent assay)-type assays, and no statistically significant changes in AR protein expression levels were observed B.Visualization of the localization of AR protein expression in U87 cells cultured under different test conditions.AR protein expression was also checked by confocal microscopy. Images of the cultures, carried out under four different culture conditions , were taken using confocal microscopy after incubation with an AR antibody with a fluorescent dye are shown in The quantitative analysis of the results also showed no differences between the analyzed test conditions. The intensity of expression for individual images of the given test conditions is similar B. The siAR mRNA expression levels in individual tumor structures did not differ between genders , as well as between the tumor core and peritumoral area (p = 0.0056848). In both cases, expression was lower in the tumor core than in the enhancing tumor region and peritumoral area (p = 0.0179611)  B.The immunohistochemical results confirmed the results of the protein expression studies by ELISA. The scrapings used for analysis were obtained from a paraffin block made for histopathological diagnosis, the result of which confirmed GBM. The microphotographs show features characteristic of GBM. In In the necrotic area, on average, the AR-positive cells accounted for 2–3%; in the enhancing tumor region, 90–95% in men and 85–95% in women; in the peritumoral area, 75–85% in men and 90–95% in women.AR mRNA expression was higher under nutrient-deficient conditions than in the control and lower under hypoxia than in the control.In vitro In cell cultures, there were no differences in AR protein expression between the various test conditions.AR mRNA expression did not differ between different tumor regions taken from patients .In all patients (women and men), AR protein expression was higher in the enhancing tumor region and in the peritumoral area than in the tumor core.In women, AR protein expression was higher in the peritumoral area than in the tumor core.AR gene and AR protein.There were no significant differences between men and women in the expression of the AR/AR expression in GBM and other cancers has already been the focus of many studies. Primary GBM tumors have so far been shown to express AR .,55.54,55A neuronavigation-guided biopsy recovered the glioblastoma structure in the sample tissue, so it was then followed by a craniotomy and tumor resection. The patient was discharged 9 days post operation with a recommendation of radiotherapy and chemotherapy.A culture of human brain tumor cells  was used in an in vitro model to investigate the effect of the tumor microenvironment on AR expression. The cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC). Cells were cultured in a standard medium, the composition of which is shown in 2. Cells of the U87 line were seeded into a 6-well plate  in a standard medium at a density of 20,000/cm2 and then cultured for 72 h to obtain adequate confluence in each culture well (70–80%). The old culture medium was then pulled off, and the cells were gently washed three times with warm (37 °C) PBS solution . In the next step, a specially prepared test media  was obtained by detaching the cells from the medium using trypsin . The cells were then centrifuged , the supernatant was discarded, and the cells were washed with warm PBS solution and centrifuged again . The resulting cell pellets were frozen at −80 °C for further analysis.Cultures for analysis by confocal microscopy were undertaken in 6-well plates containing slides coated with sterile-filtered 0.01% poly-l-lysine solution . After incubation with the test media, and the cells were washed with warm PBS solution and then flooded with 4% buffered formaldehyde solution, pH 6.9  then incubated for 10 min at room temperature to fix the slides. Further, the formalin was sequentially withdrawn and the cells were washed three times with warm PBS solution, then dried under a laminar chamber and secured for confocal and light microscopy analysis.AR, VEGF, CASP3, qRT-PCR analysis was performed. For this purpose, mRNA was isolated from the test material using an RNeasy Lipid Tissue Mini Kit  for tissue samples of 50–100 mg and using an RNeasy Mini Kit  for 2 × 106 U-87 line cells. The procedure was performed according to the manufacturer’s instructions, and the purity and concentration of the isolated mRNA were determined using a Nanodrop ND–1000 . In a further step, Reverse Transcription PCR was performed. For this purpose, a set of FirstStrand and oligo-dT primers  were used. The expression of the genes under study was determined using a Power SYBR Green PCR Master Mix reagent kit  and analyzed using an ABI 7500 . The primer sequences used were AR: CCAGGGACCATGTTTTGCC|CGAAGACGACAAGATGGACAA, VEGF: ACGAGGGCCTGGAGTGTG|CCGCATAATCTGCATGGTGAT, CASP3: CATGGAAGCGAATCAATGGA|CTGTACCAGACCGAGATGTC. The thermal reaction profile used in the analysis was 95 °C (15 s), 40 cycles of 95 °C (15 s), and 60 °C (60 s). The measurement was repeated twice for each sample analyzed, and the average Ct values were used for further analysis. Gene expression levels in the sample were normalized to those of an endogenous control: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The reference gene was selected based on literature data [To determine the relative expression of ure data ,59. The In order to carry out protein analyses in the tested material, homogenization was carried out, the course of which varied depending on the processed material.3 of tissue in 1 mL of cold PBS   containing proteinase inhibitors . The entire process was carried out at a temperature of about 4 °C. The resulting material was subjected to sonication.The procedure for patient-derived tissues involved knife homogenization of approximately 1 cm6 cells was knife homogenized in 0.5 mL commercial RIPA  containing proteinase inhibitors  according to the manufacturer’s instructions. The material processed in this way was then given a freezing and thawing process.Material derived from U-87 cell line cultures in pellet form containing 2 × 10In the next step, samples from patients and those obtained from cell culture were centrifuged (5 min 5000 G) to obtain the supernatant. In a further step, the determination of total protein concentration in the supernatant was performed using MicroBCAPierce™ . Samples were then prepared so that the concentration of total protein did not exceed 0.3 mg/mL. AR protein expression was determined using a commercial Human AR ELISA Kit . The results were read using a plate reader . The obtained values were converted to the amount of total protein. The analysis was carried out on material from patients in three GBM tumor structures and on material from a cell line cultured under four types of culture conditions.Tumor fragments were fixed for 24 h in a 4% formaldehyde solution after collection. The tumor tissues were then dehydrated and embedded in paraffin blocks.For further study, the blocks were cut using a microtome , and the resulting sections were placed on polylysine-coated basal slides . The slides were deparaffinized and hydrated. They were then boiled twice in 10 mM citrate buffer, pH 9.0 , for 4 and 3 min in a microwave oven (700 W). The slides were then cooled and washed with PBS . Endogenous peroxidase was blocked using a peroxidase blocking reagent from Dako LSAB + System-HRP kit  at room temperature for 10 min. In a further step, the scrapings were incubated overnight with AR Antibody . The concentration of AR Antibody was 1:50, according to the manufacturer’s recommendations. The slides were then stained with avidin-biotin-horseradish peroxidase with diaminobenzidine as a chromogen using a Dako LSAB + System-HRP kit . The slides were also stained with hematoxylin. Photographs of the slides were taken using a Leica DM5000 B  light microscope integrated with a camera. Analyses were performed for the AR receptor in sample slides made from GBM tumor sections.In order to standardize the results, a quantitative analysis was performed. In each analyzed specimen, three photomicrographs in the representative places  were taken. All cells visible in the field of view in each region were counted, and the number of AR-positive cells was calculated as a percentage of all cells/as a percentage composition. The samples were independently examined by two experienced histologists. All discrepancies were discussed right after the examination, problematic issues were resolved, and representative images were chosen.Fixed cell culture slides were washed with PBS solution , and cells were permeabilized in 0.5% TRITON ×100 solution  for 20 min. Then, the detergent was washed off with PBS, and the slides were incubated with blocking serum  for 20 min at room temperature. The slides were then incubated with AR Antibody I-strand  at the appropriate dilution for 1 h at room temperature in a humid chamber. In a further step, the I-row antibody was washed with PBS solution. The cells were then incubated with fluorochrome-conjugated II-row antibody  for 1 h at room temperature in a humid chamber in the dark in order to visualize it in the cells. Slides were again rinsed in PBS and incubated with DAPI  for 20 min at room temperature to visualize cell nuclei. After a final rinse in PBS, they were sealed in a fluorescence mounting medium . The slides were microscopically evaluated using an FV1000 confocal microscope  combined with an IX81 inverted microscope . Images were recorded using a 488 nm laser for FITC and a 405 nm laser diode for DAPI. Analyses were performed for AR receptors under 4 test conditions .In order to quantify the obtained results, the Lambda Stack analysis was performed, consisting of counting the number of pixels with given fluorescence intensity. Based on the obtained data, the total fluorescence intensity in a given image was calculated. For this purpose, the fluorescence intensity was multiplied by the number of pixels present at this level. The analysis included 4 photos from all test conditions.t-test was performed to compare the tested conditions with the results obtained from the controls. Patient tissue model: Shapiro–Wilk test was performed, and the results showed non-normal distributions for the most part. The Mann–Whitney U-test was used to compare identical structures between genders, and Wilcoxon signed-rank test was used to compare between structures within the same gender and regardless of gender. Values of p < 0.05 are considered statistically significant.The results of relative AR expression and AR protein expression converted to mg of total protein in the sample were statistically analyzed using Statistica software . In this study, two research models were used and analyzed separately. Cellular model: a Shapiro–Wilk test was performed, the results of which indicated normal distributions in all analyzed groups. A AR mRNA expression in GBM.In our study, we found that AR expression in GBM tumors did not differ significantly between women and men. We were the first to show differences in AR expression between different regions of the tumor in both men and women. Higher AR expression was found in the enhancing tumor region and in the peritumoral area compared to the tumor core. Therefore, we speculate that AR expression is not associated with a higher incidence of GBM in men. At the same time, tumor conditions (hypoxia and insufficient nutrient supply) may be factors with a significant impact on The cell line chosen for the experiment (U87) is different from the originally derived line, and some researchers have questioned the validity of using it in this type of research. It is a confirmed glioma line, and most of the works cited in the manuscript are based on this and other glioma lines.It should also be noted that the participating patients came from a specific and small region of Poland, and the presented analyses should be repeated on patient groups from other regions to generalize the results.The use of cobalt chloride to induce hypoxia does not fully reflect the hypoxic conditions prevailing in the tumor. Cobalt chloride produces the biochemical effects seen in hypoxia in culture cells, but the cells are not given the actual changes in oxygen tension levels.


The effect of subjective views of aging (VoA) on health outcomes is well established, yet this effect was rarely examined among older adults undergoing hospitalization or rehabilitation. Moreover, the additive effect of healthcare personnel’s ageist attitudes on treatment outcomes is unknown. Accordingly, these effects were examined within older adults hospitalized for osteoporotic fractures – a frequent late-life condition with cardinal functional implications. Study 1  found that feeling younger at admission predicted better rehabilitation outcomes. The reverse effect of functional independence at admission on subjective age at discharge was non-significant. Study 2  replicated these findings with additional VoA indices, and further demonstrated that rehabilitation outcomes were better when occupational and physical therapists reported low levels of ageist attitudes. Findings suggest that successful rehabilitation may be promoted by reducing negative VoA among patients as well as healthcare personnel.


Health literacy is an important social determinant of health and affects the ability to make decisions and take action to manage one’s health. The purpose of this study was to psychometrically examine the Arabic versions of HLS-EU-Q16 and HLS-EU-Q6 and their response patterns among Arabic-speaking persons in Sweden.By convenience sampling from a variety of settings, a total of 335 participants were invited to participate. The participants completed a self-assessment of comprehensive health literacy by answering the Ar-HLS-EU-Q16 questionnaire, also including the six items for Ar-HLS-EU-Q6. Statistical analysis was guided by The COnsensus-based Standards for the selection of health Measurement Instruments. Floor/ceiling effects, construct, structural and criterion validity, test-retest reliability and internal consistency reliability were analysed.In total, 320 participants were included in the psychometric evaluation. Mean age was 42.1 (SD 12.5), 63% (n = 199) were females and 53% (n = 169) had at least 10 years of education. No floor or ceiling effect were found for the Ar-HLS-EU-Q16 or Ar-HLS-EU-Q6. For both instruments, construct validity was confirmed in four out of five expected correlations . For Ar-HLS-EU-Q16, the principal component analysis resulted in a three-factor model with all items significantly correlating to only one factor. For Ar-HLS-EU-Q6, the principal component analysis supported a one-factor solution. Criterion validity showed poor agreement between the two questionnaires with a Cohen κ 0.58 (p < 0.001). Test-retest reliability showed a substantial agreement, Cohen’s κ for Ar-HLS-EU-Q16 and Ar-HLS-EU-Q6 were both 0.89. The internal consistency of both versions was acceptable, Cronbach alpha for Arabic-HLS-EU-Q16 was 0.91 and for Arabic-HLS-EU-Q6, 0.79. Split-half reliability was 0.95 and 0.78, respectively.The Arabic version of HLS-EU-Q16 shows good psychometric properties, validated in a Swedish setting. The findings can further inform and guide future validation studies in other settings worldwide. Furthermore, the results of the present study did not support criterion validity of Ar-HLS-EU-Q6. Health literacy is an important social determinant of health  that mor“health literacy is linked to literacy and entails people’s knowledge, motivation and competences to access, understand, appraise, and apply health information in order to make judgments and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life during the life course” [However, the fact that there are many different types and dimensions of health literacy makes it important to clearly identify the definition used and to carefully consider the choice of instrument so that the health literacy that one is interested in is the one being measured. According to Sorensen et al. (2012)  course” . This deOne of many instruments measuring CHL, which is frequently used worldwide, is the European health literacy survey questionnaire (HLS-EU-Q) , 6. The Arabic is the sixth most common language worldwide  and the The purpose of this study was to psychometrically examine the Arabic versions of HLS-EU-Q16 and HLS-EU-Q6 and their response patterns among Arabic-speaking persons in Sweden.The study had a prospective psychometric design and is a part of a larger project aiming to evaluate and subsequently measure CHL and electronic health literacy among Swedish and Arabic-speaking persons in Sweden –28. The The data collection was carried out from May to September 2019. Inclusion criteria were: 18 years of age or older, having Arabic as their mother tongue, and being present on the day of data collection. Convenience sampling was used, and the respondents were recruited by the last author visiting various arenas in three large Swedish cities, such as courses in civic orientation for newly-arrived refugees, an Arabic language school and some informal Arabic-language networks. For more details please, see Wångdahl et al. 2021 . InformaThe sample size of around 300 respondents was selected based on guidelines for psychometric testing of instruments . AccordiThe Ar-HLS-EU-Q16 consists of 16 items. Each of the items have the following four response alternatives: very difficult –difficult –easy – very easy. In the analysis an HLS-EU-Q16 index score ranging from 0 to 16 is calculated (requires response on at least 14 items). This is done by first dichotomizing the response alternatives into difficult (difficult and very difficult) and easy (easy and very easy), giving difficult the value 0 and easy the value 1, and then adding up the values of all items. Thereafter, the study population was divided into sub-groups based on CHL level. Based on the recommendations of the developer,  the threThe Ar-HLS-EU-Q6 consists of 6 items (included in Ar-HLS-EU-Q16) and the index score is calculated differently compared to the HLS-EU-Q16. First, each response alternative is coded separately , then the values of each item are added together and divided by the total number of items (requires a response on at least 5 items). This gives the HLS-EU-Q6 index score. Based on the recommendations of by the developer  and prevThe questionnaire also included demographic questions about age, biological sex, education level, country of birth, years lived in Sweden and health status. Health status was measured with the well-used and established question “How do you assess your overall health status? “ with its response options, “very poor, poor, fair, good, or very good” . ElectroThe COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) guided the choice of correct psychometric tests –38. DataConstruct validity, which describes how well the results from an instrument are consistent with a hypothesis  , was exaCriterion validity was examined by assessing the agreement between CHL levels defined by the Ar-HLS-EU-Q16 and CHL levels defined by Ar-HLS-EU-Q6 using the Cohen κ coefficient. The Cohen κ coefficient was also used to assess test-retest reliability, i.e., the agreement between the two points in time. A Cohen K coefficient value > 0.7 was considered acceptable . InternaA total of 333 respondents participated in the study, of these, 4% (n = 13) were excluded due to more than two missing answers on item level for Ar-HLS-EU-Q16 . There was no significant difference between those with a valid HLS-EU-Q16 index score and those excluded due to age (p = 0.87), biological sex (p = 0.97), educational level (p = 0.33), years in Sweden (p = 0.88) or self-perceived health (p = 0.85).The mean age of the 320 respondents included was 42.1 years (SD 12.5) and most were females . Most of the respondents were born in Syria  followed by respondents born in Iraq . The number of years the respondents lived in Sweden varied between 0 and 38 years (SD 9.6). Around half of the respondents had graduated from university  and two thirds of respondents perceived their own general health as good or very good . The Ar-HLS-EU-Q16 mean index score was 11.2 (SD 3.7), and the Ar-HLS-EU-Q6 mean index score was 2.7 (SD 0.6). A higher proportion of respondents had a sufficient HL level  according to Ar-HLS-EU-Q16 in comparison with respondents who had a sufficient HL according to Ar-HLS-EU-Q6 . The respondents’ mean Ar-eHEALS sum score was 28.2 (SD 6.1). . No other pattern of structural problems regarding difficulties in responding to certain items could be identified.All items of Ar-HLS-EU-Q16 and Ar-HLS-EU-Q6 had full variance  Table . A ceiliOn scale level, no floor or ceiling effect were found for the Ar-HLS-EU-Q16 or Ar-HLS-EU-Q6. For Ar-HLS-EU-Q16, four respondents (1%) had a minimum index score  and 41 respondents (13%) had a maximum index score . For Ar-HLS-EU-Q6, one respondent (0.3%) had a minimum index score  and 13 respondents (4.1%) had a maximum index score .Ar-HLS-EU-Q16 showed a weakly positive correlation with higher education level, higher self-perceived health, years in Sweden, a moderately positive correlation to a higher Ar-eHEALS sum score, and a strongly positive correlation to a higher Ar-HLS-EU-Q6. Ar-HLS-EU-Q6 demonstrated a weakly positive correlation to a higher education level, higher self-perceived health, years in Sweden, a moderately positive correlation to a higher Ar-eHEALS sum score and a strongly positive correlation to higher Ar-HLS-EU-Q16. No correlation was found between Ar-HLS-EU-Q16/Q6 and higher age Table .In terms of structural validity of Ar-HLS-EU-Q16, the assumptions for PCA were met with a KMO index value of 0.907 and a significant Barlett’s test of sphericity (p < 0.001). A three-factor solution was supported by the latent root criterion  and visual examination of the scree plot. The three-factor model accounted for 59% of the variance. The items that clustered together suggest that factor 1  represents “Find, understand, and process information in connection to health”; factor 2  represents “Find and process information in connection to health problems”; and factor 3 (item c and d) represents “Understand information from Healthcare Professionals” Table . For Ar-Criterion validity was assessed by examining the agreement between CHL levels defined by Ar-HLS-EU-Q16 respectively Ar-HLS-EU-Q6. This resulted in a Cohen κ of 0.58 (p < 0.001), in other words, a poor agreement between the two questionnaires.The test-retest group consisted of 37 respondents. There was a significant difference in the distribution of age between the re-test group and the total sample, where the respondents in the retest group were older. There were no significant differences in the distribution of sex, years in Sweden, educational level and self-perceived health. Thus, the demographics of the two groups were rather similar Table . Test-reThe internal consistency of both versions was acceptable. Cronbach alpha for Ar-HLS-EU-Q16 index score was 0.91 and for Ar-HLS-EU-Q6 index score 0.79. Split-half reliability according to the Spearman-Browns coefficient was 0.95 for Ar-HLS-EU-Q16 and 0.78 for HLS-EU-Q6.The aim of the present study was to examine the psychometric properties of the Arabic versions of HLS-EU-Q16 and HLS-EU-Q6 among Arabic speaking persons in Sweden. Our results indicate that the Ar-HLS-EU-Q16 has good psychometric properties, validated in a Swedish setting. However, the psychometric properties of the Ar-HLS-EU-Q6 were questionable as criterion validity could not be supported.Understand what the doctor says to you had missing responses of more than 5%. Early development and validation work of the HLS-EU- Q [Our study findings showed that all items on Ar-HLS-EU-Q16/6 had full variance, but 12 out of 16 items showed ceiling effects. One item LS-EU- Q  support LS-EU- Q . AnotherLS-EU- Q . This meIn the present study, we identified significant relationships between CHL and education level, self-perceived health, electronic health literacy and years in Sweden. Thus, we were able to confirm four of our five expected correlations , which provides evidence of construct validity of Ar-HLS-EU-Q16/6 as > 75% of the correlations correspond with the hypothesises stated in advance . In contIn the present study, the PCA yielded a defined structure of Ar-HLS-EU-Q16 with all items loading significantly (> 0.4) to only one factor. The factor structure and factor loading pattern found in our sample of Arabic speaking persons in Sweden differs from the original model , as wellIn the present study, we used HLS-EU-Q16 as our gold standard when assessing criterion validity. HLS-EU-Q16 is short form of HLS-EU-Q47, which to date does not exist in Arabic. However, items for HLS-EU-Q16 were selected using Rasch analysis in a large European sample, and its validity had been confirmed in further studies . CorrelaIn terms of test-rest reliability, our study findings showed substantial agreement between the points in time for Ar-HLS-EU-Q16/6, both regarding index scores  and for CHL levels . Additional, internal consistency for both scales was acceptable with alpha values > 0.7. Thus, score differences in our study sample were low, indicating that the instruments are stable over time and that reproducibility is high , 39. To In the present study, the Arabic versions of HLS-EU-Q16/6 were evaluated in a Swedish context. Importantly, although HLS-EU-Q16 has previously been validated among Arabic speaking persons  and usedThe following limitations should be acknowledged when interpreting the results. First, this study used a convenience sampling approach and participants were recruited from the three largest cities in Sweden. The sample might therefore not be representative of Arabic speaking persons living in rural parts of Sweden. However, recruitment was performed in several arenas aiming for a diversity in age, biological sex, length of stay in Sweden, and level of education. Second, we used self-administered paper and pen questionnaire, thus restricting participation to persons who can read and understand Arabic. Using face-to-face interviews might enhance the inclusion of persons with limited functional literacy. As discussed above, there is thus a need of feasible, reliable and valid ways to collect self-reported data that do not require the presence of a test interviewer and our findings support the psychometric properties of a self-administrated paper and pen version of Ar-HLS-EU-Q16. Third, this study did not further examine construct validity of Ar-HLS-EU-Q6 using confirmatory factor analysis or Rasch modelling. We therefore suggest future studies continue to examine the validity of Ar-HLS-EU-Q6 using complementary methodological approaches. Yet, we were able to assess stability over time for HLS-EU-Q16/6, which adds valuable information about the overall psychometric properties of the instruments.Our findings support the psychometric properties of the Ar-HLS-EU-Q16, and we can therefore recommend that it be used to evaluate CHL among Arabic speaking persons in Sweden. The findings can further inform and guide future validation studies in other settings worldwide. That said, however, the results of the present study did not support criterion validity of Ar-HLS-EU-Q6. A major concern is that the instrument shows limitations in distinguishing between CHL levels. Thus, there is a risk that a person’s CHL level is not correctly classified into inadequate, problematic, or sufficient. This presents a problem in using that instrument in assessing CHL, especially in clinical practice. However, our findings tend to indicate that the Ar-HLS-EU-Q6 is reliable and that it has stability over time. One of the benefits of using a six-item questionnaire is that it is less time consuming. We suggest that Ar-HLS-Q6 should be used for research purposes only if mean values are calculated and, when appropriate, compared either between groups of participants or over time. However, further studies are needed, to specifically establish validity and accuracy of thresholds of the HLS-EU-Q6 before widespread use in clinical practice..


To analyze the mechanism of testis-specific protein Y-encoded 1 (TSPY1) in male hepatocellular carcinoma (HCC).This experimental study was carried out at Guangxi Medical University’s First Affiliated Hospital, Guangxi, China, between January 2016 and December 2019. The expression of TSPY1, androgen receptor (AR), messenger ribonucleic acids (mRNAs), and proteins were detected by qRT-PCR and Western blotting. The co-localization and interaction of TSPY1 and AR were observed by immunofluorescence assay and co-immunoprecipitation. Hepatocellular carcinoma cells overexpressing and silencing TSPY1 were constructed, and the expression and phosphorylation levels of TSPY1, AR, and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway-related key molecules ERK1/2, p38, and JNK were also detected.The expression levels of TSPY1, AR mRNAs, and proteins were highly positively correlated in HCC cells in different metastatic potentials with a high correlation coefficient of R=0.929 and R=0.884. Testis-specific protein Y-encoded 1 and AR were then co-localized in the nucleus of HCC cells, and TSPY1 and AR can interact with each other. In addition, the expression of AR and phosphorylation of ERK1/2 were enhanced in TSPY1 overexpressed Huh7 cells. They were reduced in HCCLM3 cells with TSPY1 knockdown expression. In addition, in response to blocking MAPK/ERK signaling activity, AR was reduced in expression.These findings suggested that there was a positive correlation between TSPY1 expression and AR in male HCC cells, and high TSPY1 expression stimulates AR expression, MAPK/ERK signaling pathway may be involved in its mechanism. Worldwide, hepatocellular carcinoma (HCC) is the 6th most common cancer, among men it ranks 5th and among women it ranks 7th. In most countries, the prevalence is 2-4 times higher in males than in females, demonstrating that gender differences could be linked to the incidence of HCC. Our previous research has discovered that TSPY1 is highly expressed in male HCC and TSPY1 was associated with the proliferation and invasion of the HCC cells. Despite this, it is unknown what the molecular mechanism of TSPY1 is in male HCC.Recently, the testis-specific protein Y-encoded 1 (TSPY1) has been identified as an antigen that accelerates tumor growth. It has also been found that TSPY1 is commonly ectopically expressed in different somatic cancers under pathological conditions, including HCC. However, its specific mechanism remains unclear and needs further study.Androgen is a male hormone that is increasingly reported in male-dominated HCC. It plays a role in diverse pathological and physiological processes through binding to androgen receptor (AR). The development of HCC cells was discovered to be strengthened by overexpression of AR ex-vivo, in addition to HCC-initiating cells in-vivo. Many studies have shown that p38, JNK, and ERK, are associated with HCC occurrence and development. The activation of TSPY1 and AR in cancer is related to the MAPK pathway, and TSPY1 can increase the expression of the upstream factor Ras in the MAPK/ERK signaling pathway. Androgen receptor may be a downstream factor of the MAPK/ERK pathway in prostate cancer. Our previous research has found that cells and tissues of HCC expressed TSPY1 and AR simultaneously, while the function mechanism between them is still unclear. We hypothesized that TSPY1 can regulate AR expression in male HCC through the MAPK/ERK signaling pathway. Therefore, our study aims to discover the mechanism by which TSPY1 regulates AR expression in male HCC.In the mitogen activated protein kinase (MAPK) cascades, c-Jun N-terminal kinase (JNK) and p38 signaling pathways are mainly related to cell apoptosis, while extracellular signal-regulated kinase 1/2 (ERK1/2) is closely related to cell proliferation and differentiation.This experimental study was carried out at Guangxi Medical University’s First Affiliated Hospital, Guangxi, China, between January 2016 and December 2019. We used the search engine PubMed to search for previous relevant studies. Male HCC tissues were acquired from surgical cases at the First Affiliated Hospital of Guangxi Medical University. The Ethical Committee of the First Affiliated Hospital of Guangxi Medical University, Guangxi, China, approved the this study , which adhered to the principles of the Declaration of Helsinki.2) at 37°C, the cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum .Male HCC cell lines  were obtained from The Cell Bank of Academy of Sciences, Shanghai, China. In an incubator with 5% carbon dioxide (CO-ΔΔCt method to get the relative expression of TSPY1 and AR. Reverse transcription was used to synthesize complementary DNA (cDNA) from total RNA extracted from cultured cells using TRIzol reagent . Real-time quantitative reverse transcription PCR (qRT-PCR) was carried out with the FastStart Universal SYBR Green Master (ROX) kit  and StepOnePlus™ Real-Time PCR instrument . The internal reference was β-actin. We used the 2Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis was carried out on the total protein extracted from the cells. The bands were transferred to polyvinylidene fluoride membrane . The membrane was sealed with 5% BSA  at room temperature for one hour, and then incubated overnight with primary antibodies  at 4°C. After the wash, the secondary horseradish peroxidase-conjugated antibody was diluted at 1:10,000  and a one-hour incubation was carried out with the membrane at room temperature. The substrate for the enhanced chemiluminescence  western blot analysis was then prepared. The Bio-Rad  ChemiDoc™ XRS Gel Doc, a gel imaging system, was used for imaging and the band intensity was represented as a relative ratio.All frozen sections of HCC tissues were prepared by the Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Guangxi, China. Hepatocellular carcinoma cells were fixed with 4% paraformaldehyde  and HCC tissues were fixed with 4°C acetone  then the use of 0.5% Triton X-100  to permeate block with 10% goat serum . Testis-specific protein Y-encoded 1 was incubated with the rabbit anti-human TSPY1 antibody and the goat anti-rabbit fluorescent secondary antibody according to instructions. Androgen receptor was incubated with mouse anti-human AR antibodies and goat anti-mouse fluorescent secondary antibodies  according to instructions. Nuclear staining was carried out with 4′,6-diamidino-2-phenylindole (DAPI) . The co-localization was confirmed with a laser confocal microscope .The instructions of the immunoprecipitation kit  were followed. In brief, the proteins from the cells were immunoprecipitated by the rabbit anti-human AR antibody, and the resultant immune complexes were subjected to SDS-PAGE electrophoresis. After membrane blocking, the rabbit anti-human TSPY1 antibody and rabbit anti-human AR antibody  were used for Western blot analysis. Experimental design negative control group.2, and the control group was cultured with the same amount of serum-free medium. After 8-12 hours, the supernatant was discarded and serum containing complete DMEM was added to the culture. After 3-4 days, the transfection efficiency was detected by a fluorescence microscope. The protein and mRNA expression of TSPY1 was estimated by western blot and qRT-PCR analyses.Overexpression and silencing of TSPY1 in stable transgenic cell lines Gikein Co., Ltd  constructed a lentivirus vector for the overexpression of TSPY1 and a lentivirus vector for TSPY1-short-hairpin RNA (shRNA). The overexpression and silencing TSPY1 construction vectors were transfected into Huh7 and HCCLM3 cells. Thereafter, 1 ml of enhanced infection collusion , containing polybrene and virus stock, was added to each well. After mixing well, the cells were incubated at 37°C and 5% COp-value of <0.05.The analysis of the data was carried out using the Statistical Package for the Social Sciences, version 20.0 . Pearson’s product-moment correlation was used for correlation analysis. Two groups were compared with a T-test. Comparing between the groups was carried out by one-way analysis of variance (ANOVA). Statistical significance was determined by a p=0.025, p<0.05; p=0.023, p<0.05; The expression levels of TSPY1 and AR were positively correlated in male HCC cells. Real-time quantitative reverse transcription PCR and western blot analyses were used to estimate the expression levels of TSPY1 and AR from 6 Y chromosome-positive HCC cells with different metastatic potentials and Pearson’s correlation test was used to determine the relationship between TSPY1 and AR . In the Testis-specific protein Y-encoded 1 and AR co-localized and interacted in male HCC cells and tissues. We used immunofluorescence to detect co-localization between TSPY1 and AR in male HCC cells and tissues. Images were obtained using a laser confocal microscope. Testis-specific protein Y-encoded 1 was labeled green, while AR was labeled red, and both were distributed diffusely in the cytoplasm and nucleus. 4′,6-diamidino-2-phenylindole was used to stain the nuclei blue. Testis-specific protein Y-encoded 1 and AR showed obvious co-localization in the nucleus, as seen in yellow . To detep=0.034, p<0.05; p=0.024, p<0.05; p=0.036, p<0.05; p=0.027, p<0.05; The overexpression and silencing expression of TSPY1 could regulate the expression of AR in HCC cells. To investigate the impact of TSPY1 in the expression of AR, 2 HCC cells, Huh7, and HCCLM3 were transfected with TSPY1 cDNA lentivirus and TSPY1-shRNA lentivirus to overexpress and silence the expression of TSPY1. The transfection efficiency of Huh7 was 81.77%  and HCCLp=0.036, p<0.05), while the phosphorylation levels of JNK and p38 showed no obvious change (p=0.075). In contrast, the phosphorylation level of ERK1/2 was lower in HCCLM3 cells where the expression of TSPY1 was silenced compared to the control group , while the phosphorylation levels of p38 and JNK showed no significant change . We next examined whether the expression of AR was changed after the signaling pathway was blocked by U0126 in TSPY1 over-expressed Huh7 cells. Western blot analyses showed that the expression of AR protein decreased significantly in the TSPY1 over-expressed Huh7 cells treated with U0126  were analyzed using western blot. As shown in p=0.068; . Accordi We previously found TSPY1 to be highly expressed in hepatitis B virus-positive male HCC tissues, but low in normal male and female HCC tissues. Testis specific protein Y-linked 1 overexpression influences HCC cell growth and invasion, which is linked to androgen levels. These data suggested that TSPY1 and AR play an important role in male HCC.In recent years, the molecular mechanisms of HCC have been widely studied. However, there are still many unknowns regarding HCC, especially in terms of geography, ethnicity, and gender. Among men, HCC incidence is higher, and there is a correlation between male androgens, androgen receptors, and in HCC tissues, AR expression is abnormally high. and the correlation analysis found that the 2 were highly positively correlated; that provide a reliable theoretical basis that TSPY1 regulates the expression of AR. To verify whether TSPY1 regulates AR, TSPY1 was overexpressed and silenced in Huh7 and HCCLM3 cells, based on the results, compared to HCCLM3, we found Huh7 cells expressed more AR mRNA and protein, while HCCLM3 cells expressed less AR. Moreover, immunofluorescence co-localization assay also showed that both TSPY1 and AR co-localize in the cytoplasm and nucleus, predominantly in the nucleus. Based on these results, we hypothesized that TSPY1 binds AR in the cytoplasm, which facilitates AR entry into the nucleus, after which it binds to specific regions of DNA sequences and activates transcription of target genes. Co-immunoprecipitation experiments showed the presence of TSPY1 and AR proteins in the precipitated immune complexes by western blot, indicating an interaction between TSPY1 and AR.Based on our findings, we concluded that gene and protein expression levels of TSPY1 and AR tended to increase in hepatoma cells from non-metastatic or low-metastatic potential to high-metastatic potential. The expression trend of AR in HCC cells with different metastatic potential agrees with the results of AoJ et al, Researchers have discovered that cancers are closely related to the Ras pathway, and the major downstream factors of Ras signaling included ERK1/2. Testis specific protein Y-linked 1 was found to contribute to the up-regulation p-ERK1/2 in RAS signaling pathway. Therefore, TSPY1 may enhance the phosphorylation level of key molecules (such as Ras) by activating the related regulatory factors of the ERK1/2 signaling pathway, thereby activating this pathway. As a result of this study, it was found that the phosphorylation level of ERK1/2 was significantly increased in cells overexpressing TSPY1, while the level of ERK1/2 phosphorylation was significantly lower in cells with silenced TSPY1 expression. These findings confirmed that TSPY1 regulates the phosphorylation level of the ERK1/2 pathway. Previous studies have found that AR is an important downstream molecule of the MAPK/ERK signaling pathway in prostate cancer. Inhibition of the ERK1/2 pathway leads to low expression of AR. Based on the results of this study, TSPY1 can regulate AR expression through MAPK/ERK signaling. In addition to ERK1/2 phosphorylation, the expression of AR increased or decreased with TSPY1. Inhibition of the ERK1/2 signaling pathway also reduced AR expression in Huh7 cells overexpressing TSPY1. These results confirmed that TSPY1 regulates AR expression through the MAPK/ERK pathway in male hepatoma cells.In HCC, activation of MAPK/ERK signaling is frequent. Studies have shown that the mRNAs of RAF and ERK are overexpressed of HCC patients.We only studied the changes of signaling pathway and AR expression after overexpressing TSPY1 but not silencing TSPY1. The second limitation is that we also did not study how the MAPK/ERK pathway regulates AR expression. The third point is that we only did in vitro experiments, not in vivo experiments. Therefore, our prospects are to further investigate the regulatory relationship between TSPY1 and AR and establish a mouse model to verify their relationship in vivo.In conclusion, this study preliminarily explored the mechanism of TSPY1-mediated regulation of AR expression in male HCC and found that TSPY1 may activate the MAPK/ERK pathway to regulate AR expression. The findings provide a scientific basis for further study of the molecular mechanisms that cause and develop male HCC.


Both groups participated in an inpatient program of 21 days of rehabilitation, including land-based and aquatic exercise therapy, electrotherapy, and education. In the experimental group, a portion of land-based exercise therapy was supplemented by EMG-BF. A numeric rating scale (NRS), Knee Injury and Osteoarthritis Outcome Score (KOOS), use of mobility aids, 30 s chair stand test (CST), and timed up and go (TUG) test were used to measure outcomes. Both groups improved their functional abilities from day 1 to day 21 of rehabilitation. A higher proportion of participants did not use a walking aid (p < 0.002), and their NRS, KOOS, 30 s CST and TUG scores improved (p < 0.001). There were no significant differences between the groups in the outcomes. EMG-BF did not provide additional benefits to the conventional rehabilitation after TKA.The incidence of total knee arthroplasty (TKA) is steadily increasing worldwide. Therefore, it is crucial to develop efficient rehabilitation protocols and investigate the innovations in medical technology, which could improve rehabilitation outcomes. The aim of the study was to investigate the effect of adding electromyographic biofeedback (EMG-BF) to the conventional program of rehabilitation after TKA on quality of life, intensity of pain, and functional performance. The study was designed as a randomized controlled trial. A total of 131 patients were randomly assigned to two groups: an experimental group ( Knee osteoarthritis is one of the most frequent musculoskeletal degenerative disorders in older age. The knee joint has an important role in mobility and activities of daily life, and pain and lack of knee function greatly affect the quality of life . In advaTherefore, many older adults undergo total knee arthroplasty (TKA) to restore their ability to walk, independence, and quality of life. TKA has become one of the most common orthopedic procedures , and itsEfficient rehabilitation is crucial for restoring function following TKA . PatientDespite significant efforts to improve the outcomes after TKA, many patients following TKA experience persistent muscle weakness, functional impairment, and pain in the long term ,14,15. TElectromyographic biofeedback is an additional treatment modality that can be used to modulate muscle contraction by bringing the muscular tension to the level of consciousness . It is tThe objective of this study was to examine the effects of adding EMG-BF to the conventional program of rehabilitation after TKA, consisting of therapeutic exercise and electrotherapy, on objective and subjective functional outcomes, including pain, patient-reported measure of quality of life, lower body strength, mobility, and functional performance. This would add new evidence to the body of scientific knowledge regarding the optimal design of rehabilitation protocols after TKA and improve patients’ outcomes. If EMG-BF is found to have significant effects on patients’ recovery, then its use could be justified in postoperative rehabilitation after TKA. We hypothesized that adding EMG-BF to the conventional rehabilitation program would improve the objective and subjective functional outcomes of patients.The study was carried out as a prospective, parallel-group, single-blinded, randomized controlled trial on patients after TKA attending inpatient postoperative rehabilitation at Bizovacke Toplice Rehabilitation Hospital, Bizovac, Croatia, between November 2018 and December 2019. The study was not blinded to participants because of its nature. However, the assessors were blinded.Patients were recruited by direct contact after checking into a rehabilitation institution. Inclusion criteria were as follows: (1) patients of both genders after TKA without previous postoperative inpatient postoperative rehabilitation; (2) age between 18 and 79 years; and (3) ability to read, understand, and speak Croatian language.Exclusion criteria were: (1) patients after revision TKA, (2) patients with comorbidities that did not allow normal mobility due to other causes , (3) non-ambulatory patients before TKA where surgery was performed only for pain relief, and (4) patients not able to follow standard institutional rehabilitation protocol.Ethical approval was obtained from the Ethics Committee of the Bizovacke Toplice Rehabilitation Hospital, Bizovac, Croatia (no. 71/2018/I), and the trial was registered with The Australian New Zealand Clinical Trials Registry (ACTRN12618001782224). Participants gave their written informed consent. The trial was conducted in accordance with the Declaration of Helsinki.When patients satisfied all inclusion criteria and gave their written informed consent, they were randomly assigned into 2 groups: experimental (EG) and control (CG). We used a computerized randomization stratification procedure with the 1:1 ratio in permuted blocks of four to ensure a similar number of participants between groups. The assessors were blinded to the group allocation of participants.Before conducting the study, the sample size was calculated using G*Power Software , consideBoth groups participated in an inpatient program, which included 21 days of postoperative rehabilitation after TKA according to standard institution’s protocol, which consists of daily sessions of land-based exercise therapy, application of physical agents , group aquatic exercise, and individual education.Each of the 21 days of rehabilitation followed the same protocol, except on Sundays when patients did not receive physiotherapy. On average, there were three Sundays during the patients’ stay at the rehabilitation institution, and there were 18 days of rehabilitation consisting of 50 min of land-based exercise, 30 min of aquatic exercise, 10 min of interferential current therapy, and 10 min of electrostimulation. All interventions were performed on a daily basis, except individual education, which was performed once. The intervention was provided once per day, face to face, by a physiotherapist in a special hospital for rehabilitation. Adherence to the treatment was monitored by the physiotherapist in charge.Program of land-based exercise consisted of 20 exercises. Patients performed 7 variations of isometric exercises for thigh muscles and 13 dynamic exercises for lower limbs, which include active straight leg raise (upward and combined upward and into hip abduction) from a long-sitting position with and without elastic band, hip abduction straight leg raise (and in combination with hip flexion and extension), hip flexion in long-sitting position, leg extension in sitting position with an elastic band, hip abduction from sitting position, hip flexion from sitting position, hip flexion and leg extension exercise on a Swiss ball, and pelvic lift exercise. Isometric exercises were performed in sets of 1, with 5 repetitions with a maximal effort lasting 5 s. Dynamic exercises were performed in 1–2 sets, with 10 repetitions. Progression of the dynamic exercise was achieved with an elastic band, and during the last 6 days of rehabilitation, ankle weights (1 kg) were included.Interferential current therapy was provided using Myomed 632 device  for a duration of 10 min using symmetrical waves, phase duration of 100 µs, phase interval 0 µs, impulse frequency 80 Hz, modulation frequency 0 Hz, modulation program 1/1 s, with intensity in mA according to patient’s tolerance.Electrostimulation was provided using Myomed 632 device  for a duration of 10 min using symmetrical waves, phase duration of 500 µs, phase interval 0 µs, impulse frequency 1 Hz, modulation frequency 0 Hz, modulation program 1/1 s, with intensity in mA according to patient’s tolerance.Group aquatic exercise included 13 exercises for range of motion and strength .Individual education was mainly targeted to the long-term care of the prosthesis regarding how to return to activities of daily life, recommended and non-recommended activities, coping with postoperative pain, and prevention of blood clots by regular physical activity. It was provided by physiotherapists once, at the start of the rehabilitation, with additional explaining as necessary (if patients had questions) during further rehabilitation sessions.In the experimental group, a portion of land-based exercise therapy was biofeedback-assisted , with individually adjusted targets for muscular isometric contraction, according to the manufacturer’s instruction. Biofeedback was provided by superficial electromyography (EMG) unit, e.g., electrical activity of a muscle is registered and passed on as quantitative information (feedback) to the patient and the physiotherapist in real-time, during exercise. That way, the patients were able to monitor the strength of the contraction on screen and try to achieve adequate intensity of the muscular isometric contraction.The device was attached to the patient via three electrodes, two EMG electrodes, and one reference electrode. The EMG electrodes were placed on the muscle belly of the m. quadriceps femoris according to manufacturer’s instruction. The reference electrode was placed on the anterior portion of the tibia of the opposite leg. At the beginning of the biofeedback-assisted exercise, the patient performed maximal isometric contraction of the m. quadriceps femoris to establish the threshold, e.g., the target intensity of the muscular contraction. The sensitivity of the EMG signal was set to 200 µV to achieve good visibility to the patient, e.g., to achieve a good graphical representation of the muscle’s electrical activity on the screen of the device. After the patient performed maximal isometric contraction, this was recorded by the device.The physiotherapist in charge set the threshold, which was the value of the muscular contraction the patient should accomplish during the exercise, e.g., during periods of active muscular contraction. The patient should have contracted the muscle above the threshold, which was visible on the screen. The threshold was set to 80% of the maximal isometric contraction achieved at the start of the session. The biofeedback-assisted exercise consisted of isometric contractions of the m. quadriceps femoris for 15 min (10 s periods of contraction and 10 s periods of relaxation between contractions).The control group was given the same rehabilitation protocol except for biofeedback-assisted exercise. They performed this portion of isometric exercises without biofeedback assistance.Baseline information, taken at the initial interview on the first day of rehabilitation, included demographic data, medical history, height and body mass, side of the operated knee, level of the constraint of TKA, surgical technique and approach, place of the surgery and whether it was performed at the university hospital or general hospital, postoperative day, and mobility aid currently in use .Body mass index (BMI) was calculated according to the standard equation. Both groups had their assessments and measurements taken on their 1st and 21st day of inpatient rehabilitation. They included the self-reported functional status of the knee using Knee Injury and Osteoarthritis Outcome Score (KOOS), Croatian version LK1.0, pain intensity measured by numeric rating scale (NRS), functional status of the lower extremity using 30 s chair stand test (CST), and patients’ mobility using TUG.KOOS is a self-reported questionnaire consisting of 42 items in 5 separate subscales of pain, other symptoms, function in daily living (ADL), function in sport and recreation, and knee-related quality of life with acceptable psychometric properties ,22,23. SNRS is a reliable and valid method of measuring pain intensity . Common Thirty-second CST is measured as a number of stands where the participants are encouraged to complete as many full stands from a chair as possible within a 30 s time limit. The score is the total number of stands executed correctly within 30 s. This test provides a reasonably reliable and valid indicator of lower body strength in older adults ,26.TUG measures the time that a person needs to rise from a chair, walk 3 m, turn around, walk back to the chair, and sit down . It has Statistical analyses were performed using SPSS 25.0 . The normality of data was checked using the Shapiro–Wilk test, and the homogeneity of variances was checked with Levene’s test. Descriptive statistics were performed for all variables of interest and expressed as median and interquartile ranges.The distribution of data was not normal except for the variables of body height and body mass index, and we used the Mann–Whitney U test, Fisher’s exact test, and Chi-square test for between-group analysesp < 0.05. All the analyses included all participants for which data were available.For within-group analyses of dependant variables (pre- and post-intervention comparisons), we used Wilcoxon signed-rank test, Fisher’s exact test, and Chi-square test. Results were considered significant for p > 0.05). Both groups were comparable in terms of age, sex, body height, body mass, BMI, level of education, operated side of the body, level of the constraint of TKA, surgical technique and approach, place of the surgery, postoperative day, use of walking aid, KOOS, NRS, and functional tests at the beginning of their inpatient rehabilitation. All patients received posterior-stabilized implants using the medial parapatellar surgical approach. The alignment was mechanical in all but six (three from EG and three from CG) patients where kinematic alignment was used. The level of expertise of the surgeons exceeded 50 TKA per year.A total of 131 participants admitted to inpatient rehabilitation were finally enrolled in the trial and randomized into two groups: 67 to the EG and 64 to the CG. Fourteen participants (10.7%) dropped out of the trial, eight from the EG (11.9%), and six from the CG (9.4%) . Data frPatients in both groups spent 21(0) days hospitalized, and they had 18(0) days of active rehabilitation.p < 0.05) (p < 0.002). Likewise, 85% of the patients from the CG used walking aid on their 1st day of rehabilitation compared to 53% of them on their 21st day of rehabilitation (p < 0.001). Both groups improved their functional abilities from 1st day of rehabilitation until the 21st day of rehabilitation when final assessments were done. A higher proportion of participants did not use walking aid at the end of rehabilitation in both groups ( < 0.05) . In the p < 0.001)  . Pain sup < 0.001), on average, by two points in both groups (p < 0.001) (p < 0.001) (NRS scores lowered significantly (h groups . Both gr< 0.001) . The ave< 0.001) . In the p = 0.07) and Quality of life (p = 0.06) were borderline, there were below the determined level of significance. Additionally, there were no significant differences between groups in their 30 s CST nor TUG test results on their final assessment (sessment .This study aimed to examine the effects of adding EMG-BF to the conventional program of rehabilitation after TKA on the objective and subjective functional outcomes of the patients. The results did not confirm the beneficial effects of adding EMG-BF to the regimen of rehabilitation that consisted of therapeutic exercise performed on land and in the water, education, and electrotherapy, which consisted of interferential current therapy and electrostimulation. There were no significant differences between the EG and the CG regarding pain intensity, patient-reported measure of quality of life, or results of 30 s CST and TUG at the end of the rehabilitation. We were not able to confirm our hypothesis. To the best of our knowledge, only two previous studies have examined the effects of EMG-BF in smaller samples of the TKA population, which makes the comparison of our study with other studies difficult.EMG-BF is a relatively new method of retraining muscles in orthopedic rehabilitation. It converts myoelectrical signals in the muscle by using surface electrodes which detect a change in muscle activity and feedback this information to the user by visual or auditory signals . Its mai2, and the BMI of their patients was, on average, 26 and 25 kg/m2, respectively. In our study, the median of patients’ BMI was 30.7 kg/m2. Additionally, their intervention was different, and it included exercise training sessions performed twice per week for 4 months.One of the previous studies comparing the effects of EMG-BF on a sample of the TKA population was performed by Shanb et al. . They evAnother previous study, conducted by Wang et al. , examineSome other studies investigated the use of EMG-BF on different orthopedic conditions affecting the knee joint. Draper and Ballard  reportedWhile the use of EMG-BF seems promising in the rehabilitation of knee disorders, further work is required to justify its use in patients after TKA. Voluntary activation and strength of the m. quadriceps femoris are usually reduced after TKA and m. quadriceps femoris strength in some patients can drop up to 62% of preoperative levels from three to four weeks after surgery ,38. ThisSignificant efforts to improve patient outcomes after TKA, including implant design, patient optimization, perioperative pain management, and rehabilitation, have been undertaken . PostopeThis study has clinical relevance for patients after TKA. It confirmed that both groups had significant improvements after intensive inpatient rehabilitation. However, the use of EMG-BF did not justify the cost of the device, which could be taken into account when planning to purchase expensive equipment with often uncertain clinical benefits. Expensive equipment does not always give the best results.2 on average. Thus, we cannot generalize our results to the wider population of patients after TKA.The current study has some limitations. First, our period of intervention was only 21 days, which could be possibly too short a period to realize the full potential of EMG-BF on the muscle function. Furthermore, we only did one final assessment on the participants’ final, 21st day of hospital stay. Follow-up after an additional month could have shown some long-term effects of EMG-BF not visible on the 21st day of hospital stay. Additionally, we did not measure m. quadriceps femoris torque and voluntary activation, but we relied only on functional tests to assess the quadriceps strength and function. Quantitative measurements of m. quadriceps femoris torque and voluntary activation would add to the quality of the study. Likewise, our patients started the use of EMG-BF rather late in their rehabilitation, and their BMI was above 30 kg/mThe impact of our research will facilitate further research regarding the use of EMG-BF technology by seeking other modalities of its use that could be more clinically meaningful and efficient. Future research should aim to investigate the long-term effects of EMG-BF in various regimens, including different combinations of biofeedback-assisted isometric and dynamic exercise during longer periods. Additionally, apart from using EMG-BF only on m. quadriceps femoris, it should also be used on the m. gluteus medius since its strength is also diminished in patients after TKA.In conclusion, data suggest that EMG-BF proves no additional benefits to the rehabilitation after TKA. Conventional rehabilitation consisting of therapeutic exercise, education, and electrotherapy yielded comparable results in terms of patient-reported quality of life, pain intensity, and tests for lower body strength, mobility, and functional performance. Further studies are needed to confirm these findings.


Background: Observational evidence on the association of novel single nucleotide polymorphisms (SNPs) of androgen receptor (AR) and estrogen receptor-α . Other variants of AR were not related to male infertility. A significant association was noted between predisposition polymorphism ER- genotypes with male infertility. This significant association was not seen between ER- and the risk of idiopathic azoospermia or severe oligospermia. Heterozygote overdominance was also observed in ESR rs796065354 but not in the other variants studied.Conclusion: Pieces of evidence were found on the association of novel polymorphisms of AR and ER- with male infertility among the Iranian population. However, larger studies are warranted to confirm our findings.Key words:Male infertility, Androgen receptor, Estrogen receptor- Infertility is a universally common issue defined as a failure to achieve pregnancy after a year of unprotected intercourse (1). According to the World Health Organization report, around 60-80 million couples of reproductive age suffer from infertility worldwide (2).AR) mediates the androgen signaling in the testis. It has been pinpointed that disruption of the AR-signaling pathway results in a disturbance of maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids, and spermiation . Due to the fundamental effects of AR in spermatogenesis, various gene polymorphisms of AR concerning male infertility have been studied. Several meta-analyses have reported the association between AR gene CAG repeat length polymorphisms and male infertility (6-9). Despite these conclusions, some population-based primary studies did not find any significant associations between AR gene CAG as well as GGN repeat length polymorphisms and infertility among their male population .Although a complex network of environmental factors and genetic aberrations play a crucial role in the etiology of male infertility, the main cause of impaired spermatogenesis is unknown (3). In the case of spermatogenesis, androgen receptor . Female hormones and their receptor gene polymorphisms, in particular estrogen receptors, have been challenged in elucidating their role in male infertility (13). It should be borne in mind that similar to AR, ERs also contribute to several steps of spermatogenesis such as modulated sperm metabolism, reduced testicular size, and severe oligospermia. Besides, gene expression of ER is significantly reduced in Sertoli cell-only tests (14). Evidence has added new significant findings on the association between ER gene polymorphisms, especially p and x alleles, and increased male infertility .However, a recent local survey in southwest Iran has revealed that GGN repeat length polymorphisms are one important polymorphism leading to male infertility among the Iranian population (12). These findings have led to the hypothesis that other polymorphisms in theER gene polymorphisms and additional information is needed on the association between ER polymorphisms and male infertility. Considering the high infertility rate among men, especially Iranian men, understanding the main genetic factors, in particular sex hormone-related contributors, might shed light on the cause and mechanisms of male infertility which can be used in therapeutic strategies. We are aware of the lack of data on various hormonal gene polymorphisms and the risk of male infertility. Therefore, it seems that other observational studies are needed to elucidate novel gene polymorphism of AR and ER- and their causative role in the risk of infertility among men.Most of the previous studies have focused on some limited AR and ER- genes with male infertility among the Iranian population.The present case-control study, therefore, examined the association of different polymorphisms of 6/ml) and their azoospermia was diagnosed and confirmed by a specialist. Participants with any identified causes were excluded from the study. All infertile cases were assessed by physical examination and all necessary hormonal and genetic tests including karyotyping, micro-deletion of the Y chromosome, and cystic fibrosis transmembrane conductance regulator mutations. The controls were 25-45 yr old and included 120 healthy men who had normal semen tests and at least 1 child at the time of the study. To determine the quality and quantity of sperm samples, semen analysis was done twice for all participants using microscopic examination methods according to the World Health Organization standard values (16).This population-based, case-control study was carried out in 120 infertile men aged 25-45 yr presenting at the infertility treatment center of Valiasr hospital, Tabriz, Iran from March 2018 to July 2019. All cases were of idiopathic azoospermia or severe oligospermia . The PCR reactions were performed with a final volume of 25 μL. PCR products were analyzed by 2% gel electrophoresis using novel juice stain . Samples were re-genotyped to confirm the results.The allele-specific PCR or amplification refractory mutation system method was applied to identify the genetic polymorphisms. Genotype analysis for the ARAAR Ars137852599) were as follows: common forward primer 5'-AGT CTC TCTTCCTTCCCA ATA G-3', wild type reverse primer 5'-TCAGGCTGGTTGTTGTCG T-3', and mutant type reverse primer 5'-TCAGTCTGGTTGTTGTCG G-3'. The cycling condition for all primers was 95rs137852563) were as follows: common reverse primer 5'-GGG CAG AAA AGC ACC AGA CA-3', wild type forward primer 5'-GCT TGT ACA CGT GGT CAA GTT G-3', and mutant forward primer 5'-GCT TGT ACA CGT GGT CAA GTT A-3'. PCR conditions for both wild and mutant types were 95rs137852599, and rs137852563 polymorphisms, respectively.The ER- Ars796065354), 4 primers were used as follows: forward inner primer 5'-CTG ACCAAACGCTCTAAG AA-3', reverse inner primer 5'-ACAAAGCCAGGCTGT TCC-3', forward outer primer 5'-GCAGGGATACGAAAAGAC C-3', and reverse outer primer 5'-CTTACC TGGCACCCTCTT C-3'. The cycling condition for all primers was in this manner: for the first cycle, 95ER- Crs104893956) was amplified using forward inner primer 5'-CCAGGCAAACTTCAGATAATC- 3', reverse inner primer 5'-TTCTTACACCCTGGCGTC A-3', forward outer primer 5'-ACA CAA TTT CCC CTC AAG G- 3', and reverse outer primer 5'-TCTCTTAGGATCTGCTCATAG G-3' with the cycling condition 95rs104893956 polymorphism PCR products were 443, 252, and 230 bp for forwarding outer and reverse outer, forward inner and reverse outer, and reverse inner and forward outer, respectively. The PCR products of the rs104893956 polymorphism were as follows: 345, 213, and 169 bp for outer forward and outer reverse, inner forward and outer reverse, and outer forward and inner reverse, respectively.To amplify the This study was conducted following the ethical principles, the national norms, relevant guidelines, and regulations for conducting medical research in Iran. Written informed consent was obtained from all eligible subjects. The project was approved by the Science and Research Branch of Tehran's Islamic Azad University's Ethical Committee, Tehran, Iran (Code: IR.IAU.SRB.REC.1398.001).AR and ESR with male infertility was assessed by using conditional logistic regression. Statistical analyses were carried out using SPSS version 22 . P-values The frequency of genotypes in the case and control groups were evaluated for Hardy-Weinberg equilibrium (HWE) and Chi-square was calculated. The association of the SNPs of The concentration of the extracted DNA was in the range of 77-247 ng/μL. The obtained OD 260/280 was in the range of 1.78-1.89.AR and ER- variants in the fertile and infertile men. A total of 3 variants for the AR gene and 2 variants for the ER- gene were identified. There were no significant differences between the 2 groups in terms of ARrs1378525568 and rs137852563 variants and ER- gene rs104893956; however, there was a significant difference between cases and controls in terms of ER-rs796065354 (p = 0.001) and AR rs137852599 (p = 0.040).Table I shows the genotypic frequencies of AR and ER- polymorphisms with male infertility are shown in table II. We observed a significant association between AR rs1378525568 TT genotype and odds of idiopathic azoospermiaor severe oligospermia compared with normal homozygote . The HWE examination test revealed that this equilibrium for AR rs1378525568  and the additive pattern was considered. To examine whether HWE in a group was disrupted or not, the calculated Chi-square was compared to the statistics table Chi-square. If the calculated Chi-square was bigger than the statistics table Chi-square (3.8), the H0 hypothesis was rejected, which would mean HWE was disrupted in that group, and otherwise the H1 hypothesis was rejected, which would mean the group was in HWE. In terms of AR rs137852599, there was a significant association between the C allele  and odds of male infertility compared with the dominant allele . Men with this polymorphism were at a 45% decreased risk of infertility. The examination of HWE for AR rs137852599 . Therefore, statistical analysis was performed based on the multiplicative pattern. There were no significant associations between AR rs137852563 genotypes and infertility risk among men compared to the normal homozygote. For rs137852563 ; therefore the additive model was used for statistical analysis.The associations of ER-genotypes  and the risk of idiopathic azoospermiaor severe oligospermia compared to the normal homozygote. ForER- , which showed the additive pattern. Heterozygote over dominance was also observed in this SNP. In terms of ER-, there was no significant relationship between any genotypes of CT/CC, TT, CT+TT, and CT/TT+CC and male infertility compared with the normal homozygote. The HWE examination test for ER-  and the additive pattern was considered.The results showed a significant association between theAR as one of the interesting receptors has been studied frequently and its role in spermatogenesis, the survival of germ cells, and their maturation is well-defined .Due to the fundamental effects of genetic contributors on the etiology of infertility, examining the SNPs is of high priority. Among the genes involving the fertilization process, in particular, in men, hormone-related genes and their receptors due to multifunctional properties in male physiology and spermatogenesis are strongly targeted in various infertility studies. AR rs1378525568 TT genotype and AR rs137852599 C allele have a protective effect on fertility and may reduce idiopathic azoospermia or severe oligospermia risk. We also observed an association between ER- genotypes  and odds of infertility among men. However, the results failed to find any significant association between other polymorphisms of AR and ER-genes with the risk of infertility among our study population. To the best of our knowledge, this study is among the first case-control studies examining the association between specific polymorphisms of AR and ER-genes with the risk of male infertility among the Iranian population.The results of this population-based, case-control study suggested that AR is associated with male infertility (6-9). However, the findings of an up-to-date meta-analysis which were classified by region and sub-types of male infertility were inconsistent. It seems that Caucasian populations are more susceptible to this polymorphism due to AR dysfunctions (7-9). We found for the first time, an inverse association between AR rs1378525568 TT genotype and AR rs137852599 C allele with male infertility. Most previous studies have focused on CAG repeat length polymorphisms, and their results are in contrast to our findings . Besides, some studies failed to find any significant relationship between AR polymorphisms and the odds of various sub-types of male infertility (20). However, similar to other reports, we failed to find a significant association between other alleles of AR rs1378525568 as well as AR rs137852599 and AR rs137852563 variants, and the fertilization ability of men. It should be mentioned that previous data on AR polymorphisms have been limited to the CAG repeat length polymorphism, so previous findings differ from ours which has studied novel polymorphisms. Moreover, some basic differences concerning environmental factors, study population, and sample size might contribute to the conflicting findings between studies. Overall, it is suggested that further studies are required to investigate the relationship between different types of AR polymorphisms and male reproductive function.Most meta-analysis studies have concluded that the increase in CAG repeat length in ERs; including ER-, and ER-) being conventionally regarded as female hormones, there is a wide range of investigation showing their profound effects on the male reproductive system (3). In the case of infertility, the exact physiological role of estrogens in spermatogenesis is not clearly understood. However, based on recent studies, they are regarded as a “survival factor”. According to this concept, the absence of ER- results in reducing the epididymis sperm content, sperm motility, and fertilizing ability . It seems that ER- confers a stronger estrogen effect. We found, as the first case-control study, ER- genotypes  might increase the odds of male infertility. Similar to AR, ER- gene polymorphisms investigated in previous observational studies were different from the polymorphisms we studied. However, it has been reported that the (TA)ER- gene are associated with the risk of male infertility . ER- gene polymorphisms might affect sperm quality in men. ER- 397T/T and ER- 351A/A genotypes were associated with lower sperm motility in men with oligozoospermia (24). Semen variables including sperm count, motility, velocity, and morphology as well as sperm acrosin activity were significantly higher among infertile oligoasthenoteratozoospermia men who had pp and xx genotypes (15). Nevertheless, we found no significant relationship between ER- genotype and idiopathic azoospermia or severe oligospermia among our study population. It should be noted that our studied polymorphisms were completely different from other studies; therefore, this may lead to some inconsistency. Also, some controversial findings might be a reflection of the difference in study design and population. Similar to AR, it is suggested to conduct more observational studies to elucidate the association between different types of ER- genotypes with male infertility.Despite estrogens and their receptors . Such classic genomic action was considered for ER- where translocation of the ER-The exact mechanisms through which AR and ER-with odds of male reproductive function, as well as the age-matched design that led to control of potential age-dependent confounders, are the strengths of the present study. However, some limitations should be considered in the interpretation of our findings. Control genotypes should be in HWE, as long as the population they are selected from is random and large in size. A significant result showing that controls are not in HWE could arise because of 1) random chance (one of every 20 markers tested will give a p-value Being the first report on the association of novel polymorphisms of Environmental factors, smoking, high-risk jobs, residence in high-risk areas, etc. are some confounders that were not included in this study. Therefore, the generalizability of the findings should be done cautiously.AR and ER- and male infertility among the Iranian population. Based on the results, AR rs1378525568 TT genotype and AR rs137852599 C allele may have a protective effect on male fertility. In contrast, there was a significant association between predisposition polymorphism ESR- and male infertility that may be considered as a biomarker for male infertility. However, further studies with different ethnic populations and larger samples are needed to validate the findings.In conclusion, we found evidence of an association between novel polymorphisms of The authors are thankful to Dr. Danaei for her help in sampling and medical diagnosis, and to Ms. Beikzadeh for data analysis. This research received no specific grant from any funding agency in the public, commercial, or not-profit sectors.The authors declare that there is no conflict of interest.