BackgroundThe microRNA‐371a‐3p (miR‐371a‐3p) has been reported to be an informative liquid biopsy (serum and plasma) molecular biomarker for both diagnosis and follow‐up of patients with a malignant (testicular) germ cell tumor ((T)GCT). It is expressed in all histological cancer elements, with the exception of mature teratoma. However, normal testis, semen, and serum of males with a disrupted testicular integrity without a TGCT may contain miR‐371a‐3p levels above threshold, of which the cellular origin is unknown.ObjectivesTherefore, a series of relevant tissues (frozen and formalin‐fixed paraffin‐embedded (FFPE), when available) from the complete male urogenital tract (i.e., kidney to urethra and testis to urethra) and semen was investigated for miR‐371a‐3p levels using targeted quantitative RT‐PCR (qRT‐PCR).Materials and methodsIn total, semen of males with normospermia (n = 11) and oligospermia (n = 3) was investigated, as well as 88 samples derived from 32 different patients. The samples represented one set of tissues related to the entire male urogenital tract (11 anatomical locations), three sets for 10 locations, and four sets for six locations.ResultsAll testis parenchyma (n = 17) cases showed low miR‐371a‐3p levels. Eight out of 14 (57%) semen samples showed detectable miR‐371a‐3p levels, irrespective of the amount of motile spermatozoa, but related to sperm concentration and matched Johnsen score (Spearman's rho correlation coefficient 0.849 and 0.871, p = 0.000, respectively). In all other tissues investigated, miR‐371a‐3p could not be detected.DiscussionThis study demonstrates that the miR‐371a‐3p in healthy adult males is solely derived from the germ cell compartment.ConclusionsThe observation is important in the context of applying miR‐371a‐3p as molecular liquid biopsy biomarker for diagnosis and follow‐up of patients with malignant (T)GCT. Moreover, miR‐371a‐3p might be an informative seminal biomarker for testicular germ cell composition.

Lymphatic filariasis is very rare in Greece, despite her geographical position. Only a few cases about affected young men and children have been published during the last century. We report a 2-year-old Caucasian boy presented to our emergency department with a painless swelling in the right scrotum during the last 10 days. Neither fever nor discomfort was reported. In the clinical examination, the right scrotum was hard but painless. An ultrasound and a computed tomography (CT) scan showed a mass (d 2.04 x 1.34x 2.48 cm) in the right epididymis and it was thought to be a solid tumor of epididymis. A decision of an exploratory scrotal surgery was made and a right orchiectomy was performed. The histological examination showed that here were a lot of eosinophil, lymphocytes and some parasites, namely Wuchereria bancrofti in the parenchyma of both testis and epididymis. Then some more specific blood tests were done, which were negative for filariasis, and it was decided that chemotherapy was not needed. In a follow-up period of one and two years after the surgery, all the tests (blood tests and CT scan) were normal and the child had a normal everyday life. Sometimes some clinical cases in children could be tricky. In Europe, such parasitic diseases, as filariasis, are very rare and their diagnosis is difficult for even an experienced pediatric surgeon.

Polyorchidism is a very rare anomaly that is defined by the presence ofmore than two testes. Knowledge about the condition and accurate imaging diagnosis andcharacterization is important to decide the optimal line of management and avoid unnecessarysurgery. A 14-year-old male reported for Scrotal Color Doppler Ultrasound with complaint ofa painless mass in the left scrotum with no history of trauma. On physical examination, therewere two palpable, ovoid, mobile, non-tender masses in the left hemiscrotum. Scrotal ColorDoppler Ultrasonography (USG) revealed a well circumscribed supernumerary testicle in theleft scrotum adjacent to superior pole of left testis with parenchymal echogenicity and dopplervascularity like that of normal testis parenchyma. In conclusion, diagnosis was polyorchidismwith duplication of the left testicle. The patient was advised follow-up after one-year. USG is themodality of choice to assist in the diagnosis.

MP60-18 ANTI-MULLERIAN HORMONE-TO-TESTOSTERONE RATIO IS PREDICTIVE OF POSITIVE SPERM RETRIEVAL IN MEN WITH IDIOPATHIC NON-OBSTRUCTIVE AZOOSPERMIA

The lack of clinically-reliable biomarkers makes impossible to predict sperm retrieval outcomes at testicular sperm extraction (TESE) in men with non-obstructive azoospermia (NOA), resulting in up to 50% of unnecessary surgical interventions. Clinical data, hormonal profile and histological classification of testis parenchyma from 47 white-Caucasian idiopathic NOA (iNOA) men submitted to microdissection TESE (microTESE) were analyzed. Logistic regression analyses tested potential clinical predictors of positive sperm retrieval. The predictive accuracy of all variables was evaluated using the receiver operating characteristic-derived area under the curve, and the clinical net benefit estimated by a decision-curve analysis (DCA). Overall, 23 (49%) and 24 (51%) patients were classified as positive and negative sperm retrievals at microTESE. While circulating hormones associated to a condition of primary hypogonadism did not predict sperm retrieval, levels of anti-Mullerian hormone (AMH) and the ratio AMH-to-total Testosterone (AMH/tT) achieved independent predictor status for sperm retrieval at microTESE, with a predictive accuracy of 93% and 95%. Using cutoff values of <4.62 ng/ml for AMH and <1.02 for AMH/tT, positive sperm retrieval was predicted in all individuals, with 19 men out of 47 potentially spared from surgery. DCA findings demonstrated clinical net benefit using AMH and AMH/tT for patient selection at microTESE.

Testicular cytology by fine needle aspiration in domestic cats

Spermatogonia cells are considered as a valuable genetic material to produce individuals with desired traits in the framework of preservation of animal and poultry gene pool (cryobanks) and genetic modification of their genome (transgenesis). This technology is based on the introduction of donor spermatogonia into the testes of male recipients and the following obtaining the exogenous sperm to produce progeny with desired characteristics. A key step of this technology is the removal of own spermatogenic cells (spermatogenesis inhibition) in the male recipient. We have optimized the method to inhibit the spermatogenesis in chicken testes using alkylating agent busulfan. The multiple injections of busulfan at the concentration from 40 to 100 mg per 1 kg of body weight was performed directly into the parenchyma of testes of six month old chicken males (n = 16). In order to detect the presence of spermatogenic cells in seminiferous tubules, we carried out the histological studies of testes in one month after injection. We observed the decrease in diameters and the number of spermatogenic cells at the cross sectional area of the seminiferous tubules in the experimental group as compared to the control. When using busulfan at concentration of 40 mg/kg the number of cells averaged 35±3 cells per seminiferous tubule, which was 32.8 times less as compared to the control (1149±95). At higher concentrations of busulfan the number of cells decreased and averaged 20±2, 12±0.5 and 9±0.4 cells at concentration of 60, 80 and 100 mg/kg, repectively. Analysis of cell types of the seminiferous tubules revealed that mainly the Sertoli cells and spermatogonia represented the cell population. Moreover, with increasing concentrations of busulfan the ratio of spermatogonia was significantly reduced. The ratio of Sertoli and spermatogonia cells after introduction of busulfan into the testes at a concentration of 40, 60, 80 and 100 mg/kg was 57 and 43%, 75 and 25%, 95 and 5 % and 96 and 4%, respectively. However, the application of busulfan at a concentration 100 mg/kg led to high mortality of chicken. Thus, we demonstrated that the optimal concentration of busulfan to kill the spermatogonial cells was 80 mg/kg. The complete inhibition of spermatogenesis was observed in 9-10 months after introduction of busulfan at this concentration. The study was supported by the Russian Science Foundation within Project no.16–16–10059.

The current study aimed to investigate the effects of varicocele on testis parenchyma by using ZOOMit DWI and conventional DWI and to compare both diffusion methods. Forty-five patients with unilateral varicocele diagnosed by physical examination and color Doppler ultrasound between July 2015 and December 2015 and 32 healthy volunteers were included in the study. ZOOMit and conventional DWI were performed for all patients with a 3-T MRI scanner. Apparent diffusion coefficient (ADC) values calculated using both conventional and ZOOMit DWI decreased in the patients with varicocele of the testis, when compared with the healthy volunteer control group. Furthermore, it was determined that conventional and ZOOMit ADC values for the contralateral side without varicocele also decreased when compared with the healthy volunteer control group (p < 0.05). However, there was no statistically significant difference between testes with or without varicocele in terms of conventional ADC values (p = 0.183), whereas ZOOMit ADC values for testes with varicocele were found to be statistically significantly lower than those for testes without varicocele (p < 0.05). A significant negative correlation was found between venous diameter measured both at rest and during the Valsalva maneuver and ZOOMit ADC values in testes with varicocele. For patients with varicocele, conventional and ZOOMit DWI may be predictive of histopathologic changes in the testis, and ZOOMit DWI may be more effective in the diagnosis, treatment, and postoperative response in patients with varicocele when compared with conventional DWI.

The objective of this study was to investigate the toxic effects of arsenic on testicular measurements and histology of adult Teddy goats bucks and to examine whether these toxic effects are scavenged by vitamin E. Twelve adult Teddy goat bucks were divided randomly into three groups, A, B and C. Group A was kept as control, the B was given sodium arsenite 5 mg/kg BW per day, and group C was fed with vitamin E 200mg/kg BW per day+arsenic 5mg/kg BW per day. This treatment was continued for 84days. Analysis of data revealed that the testicular measurements (scrotal circumference, width, length and weight) were significantly reduced in arsenic-treated animals, whereas there were ameliorating effects of vitamin E on these parameters. The major histopathological changes were present in the form of loss of germinal epithelium, atrophy of Leydig cells and vacuolations. Vitamin E in combination with sodium arsenite increased the active spermatogenesis as well as restoration of germinal epithelium. It can be concluded from the present findings that sodium arsenite causes toxicity in the male reproductive system of Teddy goat bucks with major changes in parenchyma of testes. Supplementation of vitamin E has protective effects on the toxicity of sodium arsenite on the reproductive system of male Teddy goat bucks.

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17-19%), whilst stages II to V showed the lowest frequencies (~2-4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.

Most hinnies (female donkey×male horse) and mules (female horse×male donkey) are sterile with few reports of equine fertile hybrids. The main cause of this sterility is thought to be a meiotic block to spermatogenesis and oogenesis. This study compared the developmental features of the testes and a histological analyses of spermatogenesis in a male hinny with those of a normal, fertile stallion and Jack donkey. Hinny testes showed a thicker tunica albuginea, fewer blood vessels and more connective tissue in the testis parenchyma than those of the stallion and Jack donkey. Although the mean number of seminiferous tubules was significantly higher in stallion and hinny than Jack donkey (p<0.01), the mean proportion of seminiferous tubules was lower in the hinny (p<0.01) which resulted in a smaller diameter of seminiferous tubules. The mean number of spermatogonia and spermatocytes per unit area were significantly lower in hinny testis (p<0.01) and no spermatids or mature spermatozoa cells were found during immunofluorescent analyses. These results indicated that defects in seminiferous tubule development and structure occur in the testis of hinnies. Furthermore, most spermatogonia and spermatocytes cease development in synapsis during mid-meiosis of spermatocytes, which results in a block to spermatogenesis that prevents the formation of spermatids and matured spermatozoa during meiosis in male hinnies.

Telocytes (TCs) are novel interstitial cells that have been found in various organs, but the existence of TCs in the testes has not yet been reported. The present ultrastructural and immunohistochemical study revealed the existence of TCs and differentiate these cells from the peritubular cells (Pc) in contact with the surrounding structures in the testes. Firstly, our results confirmed the existence of two cell types surrounding seminiferous tubules; these were Pc (smooth muscle like characteristics) and TCs (as an outer layer around Pc). Telocytes and their long thin prolongations called telopodes (Tps) were detected as alternations of thin segments (podomers) and thick bead-like portions (podoms), the latter of which accommodate the mitochondria and vesicles. The spindle and irregularly shaped cell bodies were observed with small amounts of cytoplasm around them. In contrast, the processes of Pc contained abundant actin filaments with focal densities, irregular spine-like outgrowths and nuclei that exhibited irregularities similar to those of smooth muscle cells. The TCs connected with each other via homocellular and heterocellular junctions with Pc, Leydig cells and blood vessels. The Tps of the vascular TCs had bands and shed more vesicles than the other TCs. Immunohistochemistry (CD34) revealed strong positive expression within the TC cell bodies and Tps. Our data confirmed the existence and the contact of TCs with their surroundings in the testes of the Chinese soft-shelled turtle Pelodiscus sinensis, which may offer new insights for understanding the function of the testes and preventing and treating testicular disorders.

Real-time shear-wave elastography (SWE) is a newly developed method which can obtain the stiffness of tissues and organs based on tracking of shear wave propagation through a structure. Several studies have demonstrated its potential in the differentiation between diseased and normal tissue in clinical practices, however the applicability to testicular disease has not been well elucidated. We investigated the feasibility and reproducibility of SWE in the detection of testicular torsion. This prospective study comprised 15 patients with complete testicular torsion. Results obtained from SWE along with conventional gray-scale and color Doppler sonography and post-operative pathology were compared. The results revealed that (i) the size of injured testis was increased and the twisted testis parenchyma was heterogeneous. The blood flow signals in injured testis were barely visible or absent; (ii) The Young’s modulus, including Emean, Emax, Emin and SD values in the border area of torsional testis were higher than those of normal testis (Emean, 78.07±9.01kPa vs 22.0±5.10kPa; Emax,94.07±6.53kPa vs 27.87±5.78kPa; Emin, 60.73±7.84 kPa vs 18.90±4.39kPa; SD, 7.67±0.60 kPa vs 2.30±0.36 kPa, [P<0.05]); The Emax and SD values in the central area of the torsional testis were higher than the corresponding area of the normal testis (Emax, 8.23±0.30 kPa vs 3.97±0.95kPa; SD, 1.5±0.26kPa vs 0.67±0.35kPa,[P<0.05]) and Emin values was lower than those of normal testicles(0.93±0.51kPa vs 1.6±0.36kPa; [P<0.05]); (iii) The Young's modulus measurement between two physicians showed good agreement. The pathological findings were accordance with SWE measurement. SWE is a non-invasive, convenient and high reproducible method and may serve as an important alternative tool in the diagnosis and monitoring the progression of the acute scrotums, in additional to conventional Doppler sonography.

Increased chromatin fragmentation and reduced acrosome integrity in spermatozoa of red deer from lead polluted sites

To determine a paradigm that will be helpful for urologists to manage fibrous pseudotumors, which are a very rare condition of the testis. We retrospectively evaluated the patients with fibrous pseudotumors in our uropathological database from 1995 to 2013. Patients who had tumor markers and ultrosonography (USG) screening before surgery and a final pathology report of a fibrous pseudotumor were included in the study. In total, 838 patients with a testis mass were evaluated. Only 6 of these patients met the inclusion criteria. The mean age was 34 years (min: 20, max: 72). Serum tumor markers were in the normal range for all patients. The scrotal tumors were not clearly related to the testis parenchyma, and the radiologists could not definitively determine the nature of the masses (benign or malignant). A concomitant hydrocele was detected in 2 patients. After inguinal exploration, radical orchiectomy was performed in one patient with an ipsilateral atrophic testis, and biopsies were taken from the lesions for frozen section analysis in the other 5 patients. Pathologists reported benign tumors for all of these patients based on the frozen sections, and testicular sparing surgery was then performed in these 5 patients. If scrotal tumors are detected by ultrasonography in patients with normal tumor markers, and the tumor cannot be clear distinguished from the testis, these patients might have a fibrous pseudotumor, and organ-sparing surgery can be performed on these patients.

Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.

This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42weeks of age, 15 rams were subjected to semen collection and, 1week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5±0.5cm and 109.5±6.0g, respectively. Seminiferous tubule (ST) diameter was 188.3±4.0μm and each testis contained 1.9±0.1 Sertoli cells (×10(9) ). Each Sertoli cell supported 0.1±0.01 A spermatogonia, 3.0±0.2 pachytene spermatocytes and 7.7±0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6×10(6) cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9±9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology.

The aim of this study was to evaluate the testicular lesions and their effects on the epididymal sperm parameters in the Iranian river buffalo (Bubalus bubalis). Total numbers of 117 scrota from the pubertal buffalo were provided from the local slaughterhouse. The samples were evaluated for morphological parameters and any macro- or microscopic lesions. The sterile swabs from the testis parenchyma were subjected to microbiology culture. The epididymal spermatozoon was analysed for concentration, progressive motility and abnormalities. The results showed 34.2% fibrotic adhesions between parietal and visceral layers of tunica vaginalis that was significantly different among seasons (P<0.05). The cases of unilateral cryptorchidism and bilateral Sertoli cell tumour were detected, with no spermatozoa in the respected epididymides. Microscopic examination showed 13.25% (31/234) lesions including general (51.61%; 16/31) and multifocal (29.03%; 9/31) degenerations as well as interstitial orchitis (9.68%; 3/31) and the Sertoli cell tumour (6.45%; 2/31). No relationship between the lesions and the bacterial isolation (n=6) was detected. The sperm parameters and morphological parameters of the testis were under influence of microscopic lesions (P<0.05). In conclusion, the testicular macro- and microscopic lesions may have a noticeable contribution in the Iranian buffalo fertility.

Testis morphometry and kinetics of spermatogenesis in the feral pig (Sus scrofa)

The aim of this study was to observe the prevalence of testicular microlithiasis (TM) in surgically corrected acquired undescended testis (UDT). The prevalence of TM was assessed by ultrasound. Boys and young men who had undergone orchidopexy (ORP) for acquired UDT in mid or late childhood were observed to study the long-term testicular volume. During this examination, the presence or absence of TM was also assessed. TM was defined as echogenic foci without shadowing within the testis parenchyma. We included 106 patients who had undergone ORP at the Medical Center Alkmaar (1986-1999) and 155 patients who had undergone ORP at the Juliana Children's Hospital (1996-2009). The majority of patients were white, Caucasian (82%). The median age at follow-up, 25.8years (range 14.0-31.6years) was higher in Medical Center Alkmaar than in Juliana Children's Hospital 13.4years (range 5.1-26.6years). From 2009 to 2011, these 261 patients (median age 18.9years) underwent an ultrasound examination. Median follow-up after ORP was 11.3years (range 1.4-23.5years); age at ORP ranged from 2.1 to 16.2years, with a median of 8.5years. TM was found in 17 (6.5%) patients (median age at follow-up 20.4years; range 11-28). No significant association was found with the incidence of TM and the operated testis, the age at ORP or the racial variance (p>0.05). ORP at diagnosis for acquired UDT is associated with a 6.5% prevalence of TM in boys and young adults.