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Introduction: Several studies assessed the sensitivity and specificity of plasma concentrations of hormones such as FSH and AMH as biomarkers that can predict the presence of sperm cells, prior to testicular sperm extraction (TESE) in azoospermic men. However, the literature contains conflicting reports in this context. Objective: To evaluate the potential of hormonal profile as a non-invasive predictor of mature sperm cells' presence in the testis of azoospermic men, and to assess if the source of these hormones- testicular or peripheral blood makes any difference. Methods: Clinical data, hormonal profile, and histological classification of 57 azoospermic men were analyzed. They were categorized into 4 following groups by their testicular pathologies: obstructive azoospermia, hypospermatogenesis, spermatocyte maturation arrest, and Sertoli cells only. The level of hormones was measured by the enzyme-linked immunosorbent assay. Statistical analyses tested the predictability of hormone values for sperm retrieval. Results: Overall, 28 patients (49%) had spermatozoa detected. FSH and Inhibin B measurements were statistically significantly different between the 4 groups with different testicular pathologies (p<0.001). AMH, FSH and Inhibin B were found to be independent predictor for positive sperm retrieval (p<0.05). However, a decision tree with random forest classifying the predictive ability of the variables to indicate the presence of sperm produced low accuracy. There was no significant difference between the hormone levels of peripheral blood source and testicular blood source hormone levels. Conclusions: AMH, FSH, and Inhibin B are optional markers for positive sperm retrieval in TESE. Increasing the research group may contribute to their ability to predict the presence of mature sperm cells in the testis of azoospermic men. Disclosure: No. [ABSTRACT FROM AUTHOR]

Introduction: Several studies assessed the sensitivity and specificity of plasma concentrations of hormones such as FSH and AMH as biomarkers that can predict the presence of sperm cells, prior to testicular sperm extraction (TESE) in azoospermic men. However, the literature contains conflicting reports in this context. Objective: To evaluate the potential of hormonal profile as a non-invasive predictor of mature sperm cells' presence in the testis of azoospermic men, and to assess if the source of these hormones- testicular or peripheral blood makes any difference. Methods: Clinical data, hormonal profile, and histological classification of 57 azoospermic men were analyzed. They were categorized into 4 following groups by their testicular pathologies: obstructive azoospermia, hypospermatogenesis, spermatocyte maturation arrest, and Sertoli cells only. The level of hormones was measured by the enzyme-linked immunosorbent assay. Statistical analyses tested the predictability of hormone values for sperm retrieval. Results: Overall, 28 patients (49%) had spermatozoa detected. FSH and Inhibin B measurements were statistically significantly different between the 4 groups with different testicular pathologies (p<0.001). AMH, FSH and Inhibin B were found to be independent predictor for positive sperm retrieval (p<0.05). However, a decision tree with random forest classifying the predictive ability of the variables to indicate the presence of sperm produced low accuracy. There was no significant difference between the hormone levels of peripheral blood source and testicular blood source hormone levels. Conclusions: AMH, FSH, and Inhibin B are optional markers for positive sperm retrieval in TESE. Increasing the research group may contribute to their ability to predict the presence of mature sperm cells in the testis of azoospermic men. Disclosure: No. [ABSTRACT FROM AUTHOR]

Introduction: In the present era, the extensive use of modern cell phones has raised the health risks associated with the radiofrequency-electromagnetic radiation (RF-EMR) they emit, which the human body can absorb. RF-EMR exposure can potentially harm the process of spermatogenesis by increasing oxidative stress, elevating mitochondrial reactive oxygen species (ROS), and triggering the formation of free radicals that leads to sperm DNA damage. Our previous research revealed decreased sperm motility and viability in semen samples exposed to RF-EMR through WiFi calling. Objective: To investigate the effects of WiFi radiation on genetic variants in human sperm using current-generation smartphones. Methods: Six healthy, normozoospermic men aged 25-35 provided semen samples for this study. A current generation smartphone was used in WiFi calling mode, specifically through Whatsapp voice call, to serve as the source of RF-EMR. The call duration was six hours. Semen samples were analyzed based on the 2010 WHO guidelines before and after the radiation exposure. The genomics facility at our university extracted DNA from semen samples of each participant before and after RF-EMR exposure to perform whole exome sequencing with alignment to the reference genome. After sequencing, we compared genetic variants for each participant before and after RF-EMR exposure. Results: After six hours of exposure, a significant decrease in sperm progressive motility was observed (p<0.05). Following exposure, the median number of exonic, nonsynonymous variants absent before exposure increased to 4 (IQR 3). Across the four successfully sequenced samples, we found 20 new, exonic, nonsynonymous variants after RF-EMR exposure. Significantly, male reproductive tissues expressed 12 variants, five associated with ciliary function and two related to mitochondrial function (see Table 1). Gene Chromosome Variant Location Function SPDYE11 Chr7 A > C Spermatids • Acetylcholine receptor activity • Cell cycle regulation SPDYE17 Chr7 A > C Spermatids • Protein kinase activity • Cell cycle regulation PRB2 Chr12 T > G salivary gland • Cell cycle regulation GGT2 Chr22 G > A Spermatids • Cell cycle regulation RGPD3 Chr2 C > T Spermatids • Ciliary function EPPK1 Chr8 G > A Spermatids, spermatocytes • Epithelial structure GOLGA6L4 Chr15 G > A Spermatids, spermatocytes • Ciliary function TCP11X2 ChrX T > C Spermatids • Acrosome reaction • Cilia function • Spermatid development HRNR Chr1 A > G Mitochondria • Metal ion binding POUF1 Chr13 > GCC Mitochondria • Structure & function of spermatozoa NPIPB13 Chr16 T > A Spermatids • Ciliary function • Sperm motility NBPF11 Chr1 A > C Spermatids, Sertoli cells, Leydig cells • Cell cycle regulation • Microtubule development ANKRD20A1 Chr9 T > A Spermatocytes • Ciliary function Table 1. Candidate genes with variants after six hours of phone radiation exposure Conclusions: Based on our pilot study, we observed a decrease in sperm progressive motility and an increase in potentially related genetic variants associated with sperm motility following six hours of exposure to RF-EMR. These preliminary results highlight the need for further research to investigate the effects of RF-EMR exposure on semen parameters and to determine the clinical significance of these genetic variants. Disclosure: No. [ABSTRACT FROM AUTHOR]

Introduction: The testicular microenvironment, consisting of cells including Sertoli and peritubular cells, releases paracrine factors that may regulate testicular function. In previous research, Leptin was identified as a critical paracrine factor in the testicular microenvironment. Leptin is secreted by Sertoli and peritubular cells and binds to its receptor in Leydig cells. This interaction triggers the expression of the desert hedgehog signaling pathway, which is essential for Leydig stem cell differentiation into adult Leydig cells, which are capable of producing testosterone. However, our earlier study had a limitation: the human cell model used was derived from testes biopsies, lacking an intact immune microenvironment. This study investigates the effect of Leptin on a complete testicular immune microenvironment in a murine model. Objective: To investigate the impact of Leptin on a complete testicular immune microenvironment. Methods: We used 6-week-old C57BL6 mice, exposed to two different concentrations of Leptin, 10 and 100 mg/ml, for seven days. After the treatment period, the animals were humanely euthanized, and their blood and testes were collected. Blood samples underwent a complete blood count differential profile analysis using a Hemavet Mascot Multispecies Hematology System Counter 1500R. Testes samples were subjected to comprehensive immunophenotyping, allowing us to assess the impact of Leptin treatment on various immune cell types within the testes Results: Leptin at a low dose (10 mg/ml) showed increased, but not significant, levels of serum testosterone (13.20 + 0.37 ng/dl), while LH and FSH levels significantly (p<0.05) increased. In contrast, animals receiving 100 mg/ml Leptin showed no significant changes in hormone levels. CBC profiling indicated that lymphocytes and platelets were significantly influenced, specifically with low doses of Leptin. Comprehensive immunophenotyping revealed a significant increase in CD4+ (helper T cells) and CD8+ (cytotoxic T lymphocytes) cell numbers in animals treated with low doses of Leptin, a pattern not observed with high doses. Conclusions: This study uncovers previously unexplored aspects of how paracrine factors, released by Sertoli and peritubular myoid cells, can modulate the immune microenvironment within the testis, potentially impacting testosterone production. Future research will investigate the relationship between changes in immune cell types, increased T cell numbers, and their influence on Leydig stem cell differentiation into adult Leydig cells Disclosure: No. [ABSTRACT FROM AUTHOR]

Objectives: To compare surgical outcomes and retrieval rates between conventional microsurgical-assisted testicular sperm extraction (m-Tese) AND TESE PERFORMED WITH SURGICAL LOUPES (L-TESE) IN ADULT MALES AFFECTED BY NON-OBSTRUCTIVE AZOOSPERMIA (NOA). Methods: A multicentric prospective randomized trial (Ethics Committee N°: 202/2022) was conducted starting in March 2022 including NOA-affected patients. In the present study, we present our preliminary results after the first 6 months of enrollment. Adult males with NOA without genetic alterations who agreed to participate in the study and signed the required informed consent were included. Sperm retrieval rate (SRr), intra and post-operative complication rate (according to Clavien-Dindo classification), hormonal profile trends were considered as outcomes during the follow-up period. Results: 6 patients were enrolled between March 2022 and June 2022. Median follow-up was 5 months (IQR 3-6). Median age was 40 (IQR 35-44). Preoperative median FSH was 18.3 UI/l (IQR 15 -32) and total Testosterone was 3.95 ng/mL (IQR 2.7-4.9). Overall, SRr occurred in one patient (16.6%) from both sides. In patients with a negative SRr the histopathological report highlighted a Sertoli cells only syndrome (SCOS) in 4 cases and a tubular scleroyalinosis associated with early spermatogenic arrest in the last patient. No intraoperative complications were recorded. As regards postoperative complications, no significant differences were recorded between the two surgical approaches, there was a single case of wound infection (Clavien Dindo I) following the procedure. Considering the operative L-TeSE proved to be faster than m-TeSe with a median saving of 20 minutes (IQR 14-28) for each procedure. Conclusions: According to our preliminary the two procedures may guarantee comparable outcomes in term of SRr. Additionally, L-Tese may allow to save operative time when compared to the m-Tese apparently. Conflicts of Interest: The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Sertoli-cell only syndrome (SCOS) is the severe form of non-obstructive azoospermia, in which only the Sertoli cells line the wall of seminiferous tubules. Y chromosome microdeletions are the obvious cause of SCOS in men, but conclusive and summarized data is lacking on the monogenic mutations. To systematically review published data on monogenic mutations causing SCOS. Medline was searched without publication date restriction for all the genetic association studies on SCOS by using keywords: genetics of Sertoli cell-only syndrome, genetics of azoospermia secondary to SCOS and, genes of Sertoli cell-only syndrome. Quality evaluation and data extraction were done by two authors Studies included in the systematic review were presenting original clinical data, human male cases and patients with confirmed SCOS by histopathology or MD-TESE. The genetic evidence of SCOS was included even if the reported case and genetic testing were published separately. Non-English language studies, descriptive reviews, articles of gene expression in SCOS, and articles related to the other chromosomal abnormalities as a cause of SCOS were also excluded. Any dispute on exclusion and inclusion of a study was resolved by discussion and consensus between all authors. The initial database search identified 489 articles, after evaluation according to the defined criteria, only 15 studies matched the inclusion criteria for systematic review. Articles included in the systematic review were published from 2005 to 2020. Only 13% (n=2) of articles were published before 2012. From 2012 to 2020, there have been 87% (n=13) publications. Most of the studies (66.7%, n=10) were carried out on Asian populations (Japanese population) and the rest were on Caucasians. The 15 publications included a total of 1513 cases of SCOS, originating from 7 different countries. However, 151 cases appeared in 2 articles. Extracted data show that 40% of studies exclude patients with a history of infections, seminal tract obstruction, pituitary gland disorder or any other cause of testicular damage, and 27% of studies only recruit patients with no chromosomal abnormalities. Approximately 13% of eligible studies exclude the patients based on congenital abnormalities, past cryptorchidism, orchitis, cancer and other systemic illnesses. In addition, 13% of studies included patients who were diagnosed with SCOS. Only 7% of studies include the patient with unknown cause of male infertility. In all the studies only patients with normal karyotype and without Y-chromosome microdeletions were included. In all the studies semen analysis and confirmatory tests MD-TESE or histopathology were for SCOS. Most of the studies used the technique of direct sequencing and only one study performed whole-exome sequencing. Altogether approximately 51 variations in 13 genes (USP26, RAD21L, SEPTIN12, PAPOLB, PLK4, H3t, ETV5, GILZ, SIN3A, CUL4B, DMRT1, SPATA17 and, LRWD1) were identified from which only 7 variations showed a statistically significant association, but others showed rare or no association. Comprehensive large-scale studies on monogenic causes of SCOS still lacking. Altogether approximately 51 variations in 13 genes were identified from which only 7 variations showed statistically significant association. Further studies are required to determine the monogenic causes of SCOS. Work supported by industry: no. [ABSTRACT FROM AUTHOR]

Although testosterone deficiency (TD) may be present in 1 out of 5 men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, our recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). In the present study, we hypothesized that testicular TME releases paracrine factors that modulate LSC differentiation. To further examine the TME its see if its release of paracrine factors modulates LSC differentiation. A total of 13 men with testicular failure underwent testis biopsies for sperm retrieval. Using an IRB approved protocol, about 10mg of testicular tissue from each of these men were processed for Leydig stem cell isolation, culture and characterized. Cytokine antibody array was performed to identify the paracrine factors released by TME. The cells were treated with hedgehog signaling agonist and antagonist in the presence or absence of identified paracrine factors from the conditioned media. Two independent biopsies were treated with siRNA against LEPR (25pmol) for 48hrs. This was followed by evaluating the extent of knockdown on LEPR and B3HSD. Further, to evaluate the functional aspects (cell proliferation) modulated by Leptin-DHH signalling by first sorting the Sertoli cells, Peritubular myoid cells, Leydig stem cells and Adult Leydig cells using surface markers. These cells were then treated with Hedgehog agonist (SAG) and antagonist (Vismodigib), in the presence or absence of increasing doses of Leptin (0, 1, 10 ng/ml respectively) 48 hours post treatment, these cells were used for checking the impacts of Leptin-DHH treatments on cell proliferation by MTT assay. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t-test. We successfully isolated and cultured up to 5 × 106million LSC's / biopsy from all 13 human testis biopsies. These cells were characterized for the presence or absence of different cell types in testis such as adult Leydig cells, Leydig stem cells, Sertoli cells, peritublar myoid cells and germ cells. Results showed that, of the cells cultured, up to 70% of the cells were Leydig stem cells and 10% of them were Sertoli-cell in origin on day 14. IF and qPCR data showed as the majority of cell population was undifferentiated (PDGFR-α). Upon stimulation by LH, the expression of 3βHSD (mature Leydig cells) was increased and that of PDGFR-α was decreased. Results from siRNA against LEPR has shown the down-regulated LEPR and B3HSD expression levels. The results from cell proliferation showed that there is minimal to no impact of leptin treatment on upon DHH agonist (SAG) or antagonist (Vismodigib) treatment in either cell types therefore suggesting that Leptin is not involving in the proliferation mechanism. Furthermore, the results demonstrated that TME secretes leptin which induces LSC differentiation and increases T production. Leptin's effects on LSC differentiation and T production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin acts on LSCs upstream of DHH; leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effects on leptin levels. Taken together, results from this study demonstrate the instrumental role of leptin as a paracrine factor secreted by TME on human LSC function and differentiation. Moreover, this study established the molecular events (DHH signaling) that underlie the leptin-modulated effects on LSCs. Further studies are ongoing to validate the implications of these Leptin in terms of its role in LSCs function, differentiation and survival No [ABSTRACT FROM AUTHOR]

To define the pathological findings in a series of infertile men, and to investigate their relationship with their medical records and hormonal assessment. Retrospective, observational study in which we included all infertile men with a diagnosis of azoospermia who underwent testicular diagnostic biopsy. In our center, this process is performed systematically to all men with azoospermia. We collected patient history, findings on physical examination and hormonal levels. Pathological study analyzed the presence of all cellular maturation stages, the proportion of Sertoli, germ and Leydig cells, the size of the lumen of the seminiferous tubules, the thickening of the basement membrane, the presence of hyalinization and CIS. We identified 71 samples, with a mean age of 34.9 years (SD 6.38). The histological analysis showed 54.9% (39) of Sertoli cell only syndrome (SCO), 26.9% (19) sclerosis, 21.1% (15) hypospermatogenesis, 15.5% (11) conserved spermatogenesis, 14.1% (10) germ cell maturation arrest and 7 % (5) Leydig cell hyperplasia. Of them, 32.2% (22) were mixed pattern. Mean testosterone was 16 ng/dL (SD 7.31), LH 9.18 IU/L (SD 5.67), and FSH 17.49 IU/L (SD 12). FSH was higher (18.90 vs 9.35, p=0.02) in those with abnormal histology (52) in contrast to those with normal histology (9). Higher levels of FSH were found in SCO patients (20.87 vs 12.95), being the sclerosis group the one with the highest mean FSH (26.79 vs 16.09, p=0.01). Also, significant higher LH levels in those men with abnormal histology (9.87 vs 5.29, p=0.02) was found, although when analyzing for different histologic diagnosis, no significant differences were observed. In our series, the most frequent pattern in biopsies is SCO 54,9% (39). We observed significantly elevated levels of FSH and LH in the abnormal histology group, which is consistent with what has been previously described in the literature. We do not have any conflict of interest. [ABSTRACT FROM AUTHOR]

To define the pathological findings in a series of infertile men, and to investigate their relationship with their medical records and hormonal assessment. Retrospective, observational study in which we included all infertile men with a diagnosis of azoospermia who underwent testicular diagnostic biopsy. In our center, this process is performed systematically to all men with azoospermia. We collected patient history, findings on physical examination and hormonal levels. Pathological study analyzed the presence of all cellular maturation stages, the proportion of Sertoli, germ and Leydig cells, the size of the lumen of the seminiferous tubules, the thickening of the basement membrane, the presence of hyalinization and CIS. We identified 71 samples, with a mean age of 34.9 years (SD 6.38). The histological analysis showed 54.9% (39) of Sertoli cell only syndrome (SCO), 26.9% (19) sclerosis, 21.1% (15) hypospermatogenesis, 15.5% (11) conserved spermatogenesis, 14.1% (10) germ cell maturation arrest and 7 % (5) Leydig cell hyperplasia. Of them, 32.2% (22) were mixed pattern. Mean testosterone was 16 ng/dL (SD 7.31), LH 9.18 IU/L (SD 5.67), and FSH 17.49 IU/L (SD 12). FSH was higher (18.90 vs 9.35, p=0.02) in those with abnormal histology (52) in contrast to those with normal histology (9). Higher levels of FSH were found in SCO patients (20.87 vs 12.95), being the sclerosis group the one with the highest mean FSH (26.79 vs 16.09, p=0.01). Also, significant higher LH levels in those men with abnormal histology (9.87 vs 5.29, p=0.02) was found, although when analyzing for different histologic diagnosis, no significant differences were observed. In our series, the most frequent pattern in biopsies is SCO 54,9% (39). We observed significantly elevated levels of FSH and LH in the abnormal histology group, which is consistent with what has been previously described in the literature. We do not have any conflict of interest. [ABSTRACT FROM AUTHOR]

Testicular microlithiasis is prevalent in sub-fertile men but data regarding its association with sperm retrieval rates in azoospermic men are lacking. To investigate the prevalence of testicular microlithiasis and its relationship with sperm retrieval rates and histopathology in azoospermic men. Eighty-six consecutive men underwent scrotal ultrasonography prior to microsurgical testicular sperm extraction (microTESE). Sperm retrieval rate, testicular histopathology, testicular size, reproductive hormone estimation, karyotyping, Y chromosome microdeletion analyses and the presence of varicocele and hydrocele were compared between men with and without testicular microlithiasis. In total, sperm retrieval rate was 40%. Testicular microlithiasis was detected in 12 men (14%). In this subgroup, sperm retrieval rate was only 8% (1/12) as opposed to 45% (33/74) in azoospermic men with no evidence of microlithiasis (p=0.02). Mean right and left testicular diameters were significantly lower in the microlithiasis group compared to men without microlithiasis (2.4 cm vs. 3.2 cm and 2.4 cm vs. 3.3 cm, respectively, p=0.04). There was no significant difference in any other parameter between the groups (age, FSH levels, prevalence of XXY karyotype, Y chromosome microdeletions, sonographic varicoceles or hydroceles). All eleven patients with microlithiasis and no spermatozoa found were diagnosed with Sertoli cells only on histopathology. The patient with testicular microlithiasis and successful sperm extraction was diagnosed with mixed atrophy. Among our cohort of azoospermic men who underwent scrotal ultrasound prior to microTESE, the presence of testicular microlithiasis was associated with very low sperm retrieval rates. Larger, prospective studies should be conducted to strengthen our results. No [ABSTRACT FROM AUTHOR]

Impaired testosterone production as a result of Leydig cell loss or dysfunction can occur in men with testicular failure. We have shown in our recent study conducted in murine models that, Leydig stem cell transplantation along with peritubular myoid cells and Sertoli cells could be used to physiologically increase serum testosterone thereby potentially minimizing the adverse effects. [Extracted from the article]