Normal Testis Research Articles

Shear Wave Elastography Evaluation of Testes in Patients With Varicocele.

We aimed to determine histological damage in patients with varicocele by comparing the elasticity of their affected testes with that of their normal contralateral testes as well as normal testes of control subjects without varicocele using shear wave elastography (SWE). In total, 48 patients with varicocele (96 testes) and 52 control subjects (104 testes) were included in this study. Shear wave elastography values were measured in the longitudinal plane using an oval region of interest that covered as much of the testicular contours as possible. Fifty testes with varicocele were classified as group A, 46 normal contralateral testes of the same patients as group B, and 104 normal testes of control subjects as group C. The normal group C testes were randomly chosen from patients who had applied for ultrasonography for any reason and agreed to participate in the study. The testicular volume and SWE values were compared between these 3 groups. The average age of the patients was 28.45 years, and no significant difference in age was found between the 3 groups (P = 0.665). A significant difference in the testicular volume was found between groups A and C (P = 0.014). The SWE values were significantly higher in group A than in groups B and C (P < 0.001). No significant correlation was observed between the testicular volume and SWE values in any of the groups. Our results showed that SWE can be used as an effective technique to assess testicular stiffness in patients with varicocele to predict interstitial fibrosis and the severity of histological damage.

Human amniotic fluid-based exposure levels of phthalates and bisphenol A mixture reduce INSL3/RXFP2 signaling

BackgroundThe presence of chemical pollutants in the environment can affect human health. Epidemiological and in vivo experimental studies reveal reprotoxic effects (undescended testis) of phthalates (diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP)) and bisphenol A (BPA), resulting in particular of a decrease in INSL3 (Insulin-Like 3 peptide) production. This hormone is essential for normal testis development and acts on a G protein-coupled receptor: RXFP2. ObjectivesThe aim of this study was to evaluate the individual and combined impacts of DEHP, DBP, and BPA on human RXFP2 (hRXFP2) activity. MethodsWe used HEK293 cells transiently transfected with hRXFP2 and receptor activity was analyzed by measuring intracellular cAMP production. The mixture was established at concentrations reported in human amniotic fluid, for the three compounds. ResultsIndividually, DEHP, DBP and BPA increased the response to INSL3 by 19.3 to 27.5%. This potentiating effect was specific for RXFP2, because it was absent in the cells which did not express this receptor. On the other hand, and interestingly, the mixture of the three compounds reduced significantly the response to INSL3 by 12%, and the observed effects were opposite to those predicted, suggesting an antagonist effect. Discussion-ConclusionTaken together, our results demonstrate for the first time that a mixture of phthalates and BPA present in human amniotic fluid disturbs the human RXFP2 function. Moreover, we demonstrate that mixture can produce potential antagonistic effects that are not displayed by the compounds, individually.

Comparative Transcriptomics Analysis of Testicular miRNA from Cryptorchid and Normal Horses.

Simple SummaryThe testis is an important organ for mammals, and testicular microRNA expression is associated with male fertility to a certain extent. Cryptorchidism is the failure of one or both testes to descend into the scrotal sac. It is a common congenital malformation in horses. The major clinical consequence of this abnormality is impaired fertility. The expression of testicular microRNAs is influenced by many factors, including high temperature and disease, in cryptorchid horses. Here, we investigated the microRNA expression levels of normal and retained testes. Their expression patterns showed significant differences. In addition, we obtained comprehensive expression data for equine testicular microRNA, which is fundamental information for further analysis.In the biological process of testicular spermatogenesis, the expression and interaction of many genes are regulated by microRNAs (miRNAs). However, comparisons of miRNA expression between descended testes (DTs) and undescended testes (UDTs) are rarely done in horses. In this study, we selected two UDTs (CKY2b and GU4b) from Chakouyi (CKY) and Guanzhong (GU) horses and eight DTs (GU1–3, CKY1, CKY3, CKY2a, GU4a, and GU5). Three groups were compared to evaluate expression patterns of testicular miRNA in stallion testes. Group 1 compared normal CKY horses and GU horses (CKY1 and CKY3 vs. GU1–3). Group 2 (CKY2a and GU4a (DTs) vs. CKY2b and GU4b (UDTs)) and group 3 (GU1–3, CKY1, CKY3 (DTs) vs. CKY2b and GU4b (UDTs)) compared the expression levels in unilateral retained testes to normal testes. The results show that 42 miRNAs (7 upregulated and 35 downregulated) had significantly different expression levels in both comparisons. The expression levels of eca-miR-545, eca-miR-9084, eca-miR-449a, eca-miR-9024, eca-miR-9121, eca-miR-8908e, eca-miR-136, eca-miR-329b, eca-miR-370, and eca-miR-181b were further confirmed by quantitative real-time PCR assay. The target genes of differentially expressed miRNAs in three comparisons were predicted, and the functions were annotated. The putative target genes of the 42 co-differentially expressed miRNAs were annotated to 15 functional terms, including metal ion binding, GTPase activator activity, zinc ion binding, intracellular, cytoplasm, and cancer pathways, and osteoclast differentiation. Our data indicate that the differentially expressed miRNAs in undescended testis suggests a potential role in male fertility and a relationship with cryptorchidism in horses. The discovery of miRNAs in stallion testes might contribute to a new direction in the search for biomarkers of stallion fertility.

B22 Development of a Novel Serum Marker for Detecting Small-Cell Lung Cancer by Targeting a Cell Adhesion Molecule 1 (CADM1)

Small-cell lung cancer (SCLC) accounts for about 15% of lung cancer. Although SCLC often responds favorably to combined-modality chemotherapy at the initial treatment, resistant tumors develop rapidly, which makes SCLC one of the representative cancers refractory to any therapeutic approaches. Moreover, molecular targeting therapy has not been developed for SCLC so far. Therefore, novel approaches to the diagnosis and treatment of SCLC on the basis of molecular understanding would be prerequisite to control this refractory cancer. One of the most critical issues of SCLC is its early detection in the initial screening and after chemotherapy. For this purpose, progastrin-releasing peptide (ProGRP) and neuron specific enolase (NSE) are widely used for serum markers for detection of SCLC, although combination of ProGRP and NSE can detect at most 60% of SCLC. We have previously demonstrated that CADM1, a member of the immunoglobulin superfamily cell adhesion molecules, is highly expressed in around 75% of SCLC. In addition, SCLC expresses a splicing variant, CADM1v8/9, which is observed specifically in normal testis. Here, we report that the extracellular fragment of CADM1v8/9 is digested by ADM17 and released into cell medium or human serum. Then, we generated specific monoclonal antibody against CADM1v8/9 using Cadm1-deficient mice and developed a serum diagnostic marker for SCLC. Preliminary study shows that CADM1v8/9 detects 47% of SCLC, which is independent of and partly overlaps with the cases detected by ProGRP. CADM1v8/9 can also detect a significant portion of patients with limited disease of SCLC. Furthermore, the amount of CADM1v8/9 fragments correlates well with the disease activity of SCLC before and after the chemotherapy. These findings indicate that detection of CADM1v8/9 in serum from patients is a novel and promising approach to detect and follow up SCLC patients. CADM1 would also provide a promising target for the treatment of SCLC.

Plasma level of laminin 5 and collagen IV in cryptorchidism

PurposeLaminin 5 and collagen IV are the main compounds of the extracellular matrix of the germinal epithelium. The purpose of this study was to evaluate the concentration of these two markers of fibrosis in the plasma of boys with congenital unilateral cryptorchidism. Materials and methodsThe study group comprised 43 boys aged 1–3 years with congenital unilateral cryptorchidism. The control group included 54 healthy, age matched boys, admitted for planned hernioplasty. To assess laminin 5 and collagen IV in the plasma of boys with unilateral cryptorchidism, we used a new biosensor with Surface Plasmon Resonance Imaging technique detection. ResultsThe median concentration of laminin 5 and collagen IV in the serum of boys with congenital, unilateral cryptorchidism was higher than in boys with normal scrotal testis. The difference was statistically significant (p < 0.0001). We did not notice a correlation between a higher position of the testicles in the inguinal and/or their condition and levels of laminin 5 and collagen IV in the plasma. ConclusionLaminin 5 and collagen IV concentrations in the plasma were higher in patients with congenital unilateral cryptorchidism. We believe that in the future, our results could be compared with fertility level in adulthood.

ScaR-a tool for sensitive detection of known fusion transcripts: establishing prevalence of fusions in testicular germ cell tumors.

Bioinformatics tools for fusion transcript detection from RNA-sequencing data are in general developed for identification of novel fusions, which demands a high number of supporting reads and strict filters to avoid false discoveries. As our knowledge of bona fide fusion genes becomes more saturated, there is a need to establish their prevalence with high sensitivity. We present ScaR, a tool that uses a supervised scaffold realignment approach for sensitive fusion detection in RNA-seq data. ScaR detects a set of 130 synthetic fusion transcripts from simulated data at a higher sensitivity compared to established fusion finders. Applied to fusion transcripts potentially involved in testicular germ cell tumors (TGCTs), ScaR detects the fusions RCC1-ABHD12B and CLEC6A-CLEC4D in 9% and 28% of 150 TGCTs, respectively. The fusions were not detected in any of 198 normal testis tissues. Thus, we demonstrate high prevalence of RCC1-ABHD12B and CLEC6A-CLEC4D in TGCTs, and their cancer specific features. Further, we find that RCC1-ABHD12B and CLEC6A-CLEC4D are predominantly expressed in the seminoma and embryonal carcinoma histological subtypes of TGCTs, respectively. In conclusion, ScaR is useful for establishing the frequency of known and validated fusion transcripts in larger data sets and detecting clinically relevant fusion transcripts with high sensitivity.

Retiform hemangioendothelioma: An uncommon vascular neoplasm

Retiform hemangioendothelioma is a rare vascular neoplasm of intermediate grade, the diagnosis of which can be challenging. We report a case of 35-year-old man with swelling in the postauricular region. He had undergone FNAC which had revealed blood only. Microscopic examination showed narrow, arborizing, vascular channels resembling normal rete testis. Evidence of mitoses or cytological atypia were lacking. Immunohistochemistry showed diffuse and strong staining for CD34 along with CD31 positivity. Immunostains for D240 and GLUT1 were negative. A diagnosis of retiform hemangioendothelioma was made. Histologically, it should be distinguished from Kaposiform hemangioendothelioma, Dabska tumor, epithelioid hemangioendothelioma, and angiosarcoma.

Eggshell calcification in a case of longstanding hydrocele

An 80-year-old man was being evaluated for voiding luts with haematuria. On physical examination, he was found to have grossly enlarged right testis which was stony hard on palpation with...

Effects of nitric oxide on reproductive organs and related physiological processes

Nitric oxide (NO), a member of the reactive nitrogen species family, plays a role in several physiologic processes, including vasculogenesis and angiogenesis, growth and puberty, and senescence and apoptosis. NO plays an important role in the production of ovarian steroids, ovulation, and follicular apoptosis. In other words, increased activity of nitric oxide synthase (NOS) leads to an increased amount of NO, which triggers production of prostaglandins and inflammatory cascades which facilitate follicular rupture and atresia. NO concentration elevation inhibits steroid synthesis in luteal and granulosa cells. Since NO is a major paracrine mediator of various biological processes, as well as a key factor in both the reproductive cycle and embryo implantation, oversynthesis of NO in the uterus results in toxicity and inflammation in epithelial cells and immunorejection of implantation. In the male physiological system, NO synthesized by NOS plays a major role in erectile function and androgen secretion, as well as semen parameters, and oocyte junction to the sperm. Furthermore, this supposedly simple molecule is involved in a number of other functions, such as germ cell evolution, connections between sertoli cells and germ cells in the blood-testis barrier, homodynamic contraction, and germ cell apoptosis. Moreover, NO is considered a key factor in male fertility due to its widespread distribution in both normal and diseased testis tissue. The difference of expression level of NOS in normal and pathological states is a probable cause of fertility destructive processes.

The use of ex vivo ovary culture for assessment of alterations in steroidogenesis following neonatal exposure to poly(ethylene glycol)-block-polylactide methyl ether or titanium dioxide nanoparticles in Wistar rats.

Rapid development and widespread application of different types of nanoparticles (NPs) may result in increased exposure of humans and animals to NPs. Recently, reproductive toxicity due to NP exposure has become a major component of risk assessment. Current data have suggested that NPs may pose adverse effects on male and female reproductive health by altering normal testis and ovarian structure, and sex hormone levels. To detect possible alterations in steroidogenesis in adult and infantile rats following neonatal exposure to polymeric poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) or titanium dioxide (TiO2) NPs, whole ovary cultures were used. Newborn female Wistar rats were intraperitoneally (i.p.) injected daily with two different doses of PEG-b-PLA NPs (20 and 40 mg/kg body weight, b.w.) or TiO2 NPs (1% LD50 TiO2=59.2 µg/kg b.w. and 10% LD50 TiO2=592 µg/kg b.w.) from postnatal day 4 (PND 4) to PND 7. The ovaries were collected on PND73 and PND15 of PEG-b-PLA- and TiO2 NP-treated rats, respectively, and their corresponding control animals. Minced ovaries were cultured in vitro in the absence (basal conditions) or presence of gonadotropins (follicle-stimulating hormone, FSH and luteinizing hormone, LH) and insulin-like growth factor-1 (IGF-1) (stimulated conditions) for 6 days. At indicated time intervals, culture media were collected for steroid hormone (progesterone, estradiol) analysis by specific radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) kits. Basal progesterone and estradiol secretion by ovaries from adult rats (PND73) were significantly decreased (p<0.01) in both PEG-b-PLA-treated groups after 3 days and 1 day of ex vivo ovary culture, respectively, compared with control group. With the presence of FSH/LH and IGF-1 in the culture medium, progesterone and estradiol production significantly increased (p<0.001) compared to basal levels. Stimulated progesterone production was significantly decreased (p<0.05) in PEG-b-PLA40-treated group after 3 days of culture compared with controls. After ex vivo culture of rat ovaries collected on PND15, basal progesterone and estradiol levels measured in the culture media did not differ between control and both TiO2 NP-treated groups. The ovaries from rats neonatally exposed to both doses of TiO2 NPs failed to respond to FSH/IGF stimulation in progesterone secretion at all time intervals. The obtained results indicate that neonatal exposure to NPs in female rats may alter ovarian steroidogenic output (steroid hormone secretion) and thereby might subsequently induce perturbation of mammalian reproductive functions. Possible mechanisms (induction of oxidative stress, inflammation) of adverse effects of NPs on ovarian function should be further elucidated.

Long-term maternal exposure to atrazine in the drinking water reduces penis length in the tammar wallaby Macropus eugenii

Marsupials are experiencing devastating population declines across Australia. Exposure to environmental endocrine disruptors, through ingestion of contaminated resources in the environment, could be contributing to this decline. Atrazine (ATZ), a widely used herbicide in Australia, is an endocrine disruptor with the ability to cause reproductive abnormalities in a diverse range of vertebrates. We exposed adult female wallabies (Macropus eugenii) to drinking water containing ATZ (450 p.p.m) throughout pregnancy, parturition and lactation. We assessed the outcome of this exposure to the reproductive development of their young by assessing gonad and phallus development. Both these organs are especially sensitive to perturbations in the hormonal environment during development. Although no gross abnormalities were seen in gonad structure, exposure to ATZ did alter the expression of genes required for normal testis function. Furthermore, long-term exposure to ATZ resulted in a significant reduction in penis length. These results demonstrate that ATZ exposure during gestation and lactation can significantly affect the development of male young by affecting virilisation. Given the known vulnerability of macropodid marsupials to endocrine disruption, as well as their overlapping distribution with agricultural areas, these data raise major concerns for the use of pesticides in areas with fragile marsupial populations.

A Novel Tissue and Stem Cell Specific TERF1 Splice Variant Is Downregulated in Tumour Cells.

In this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged.

The effect of experimental varicocele on the apelin and APJ expressions in rat testis tissue

Varicocele, which is one of the causes of infertility in men, can be defined as the expansion of spermatic cord veins. The presence of apelin and apelin receptor (APJ) in many tissues and the effects of apelin have been reported in several studies.There is no study showing apelin and APJ protein expressions in normal and varicocele-induced testicular tissues. In this study, we aimed to demonstrate varicocele-induced changes in apelin and APJ expressions in testicular tissue by immunohistochemical and western blotting techniques.In our study, Wistar male rats were randomly divided into three groups as control, varicocele, and sham. While the control group rats were not subjected to any treatment, the unilateral varicocele model was created under anesthesia in the varicocele group. In the sham group, the left abdominal region was opened and closed to exclude the effect of the surgical procedure. At the 13th postoperative week, the left testes were obtained under anesthesia in all groups, and the immunohistochemistry and Western blotting techniques were used to detect apelin and APJ expressions.In our study; apelin and APJ were significantly expressed in control group’s testicular tissue; apelin in testicular tissues of varicocele groups increased compared to the control group, whereas APJ expression decreased.In conclusion, the presence of apelin/APJ system in normal testis and the increased expression of apelin in varicocele-induced testicular tissue suggested that apelin may have a role in the varicocele etiopathogenesis.

Tazarotene-Induced Gene 1 (TIG1) Interacts with Serine Protease Inhibitor Kazal-Type 2 (SPINK2) to Inhibit Cellular Invasion of Testicular Carcinoma Cells.

Tazarotene-induced gene 1 (TIG1) encodes a protein that is a retinoid-regulated tumor suppressor. TIG1 is expressed in most normal tissues, and downregulation of TIG1 expression in multiple cancers is caused by promoter hypermethylation. Kazal-type serine protease inhibitor-2 (SPINK2) is a serine protease inhibitor, and the SPINK protein family has been shown to inhibit the expression of urokinase-type plasminogen activator (uPA). In addition, increased levels of uPA and the uPA receptor were observed in testicular cancer tissues. This study demonstrated that TIG1 interacts with SPINK2 in NT2/D1 testicular carcinoma cells. TIG1 and SPINK2 were highly expressed in normal testis tissues, while low expression levels of TIG1 and SPINK2 were found in testicular cancer tissues. TIG1 inhibited cell invasion, migration, and epithelial–mesenchymal transition (EMT) of NT2/D1 cells. SPINK2 enhanced TIG1-regulated uPA activity and EMT suppression, while silencing SPINK2 alleviated TIG1-mediated EMT regulation, cell migration, and invasion. Therefore, the results suggest that the interaction between TIG1 and SPINK2 plays an important role in the inhibition of testicular cancer cell EMT, and suppression is mediated through downregulation of the uPA/uPAR signaling pathway.

Orexin A-Mediated Modulation of Reproductive Activities in Testis of Normal and Cryptorchid Dogs: Possible Model for Studying Relationships Between Energy Metabolism and Reproductive Control.

Orexin A (OxA) is a neuropeptide produced in the lateral hypothalamus that performs pleiotropic functions in different tissues, including involvement in energy homeostasis and reproductive neuroendocrine functions. The role of OxA is particularly important given the well-studied relationships between physiological mechanisms controlling energy balance and reproduction. The enzyme P450 aromatase (ARO) helps convert androgens to estrogens and has roles in steroidogenesis, spermatogenesis, and energy metabolism in several organs. The goal of this study was thus to investigate the role of OxA in ARO activity and the effects of this regulation on reproductive homeostasis in male gonads from healthy and cryptorchid dogs. The cryptorchidism is a specific condition characterized by altered reproductive and metabolic activities, the latter of which emerge from impaired glycolysis. OxA helps to stimulate testosterone (T) synthesis in the dog testis. We aimed to investigate OxA-mediated modulation of 17β-estradiol (17β-E) synthesis, ARO expression and metabolic indicators in testis of normal and cryptorchid dogs. Our results indicate putative effects of OxA on estrogen biosynthesis and ARO activity based on western blotting analysis and immunohistochemistry for ARO detection and in vitro tests. OxA triggered decrease in estrogen production and ARO activity inhibition; reduced ARO activity thus prevented the conversion of T to estrogens and increasing OxA-mediated synthesis of T. Furthermore, we characterized some metabolic and oxidative modulations in normal and cryptorchid dog's testis. The steroidogenic regulation by OxA and its modulation of ARO activity led us to hypothesize that OxA is a potential therapeutic target in pathological conditions associated with steroidogenic alterations and OxA possible involvement in metabolic processes in the male gonad.

Male mice lacking ADAMTS-16 are fertile but exhibit testes of reduced weight

Adamts16 encodes a disintegrin-like and metalloproteinase with thrombospondin motifs, 16, a member of a family of multi-domain, zinc-binding proteinases. ADAMTS-16 is implicated in a number of pathological conditions, including hypertension, cancer and osteoarthritis. A large number of observations, including a recent report of human ADAMTS16 variants in cases of 46,XY disorders/differences of sex development (DSD), also implicate this gene in human testis determination. We used CRISPR/Cas9 genome editing to generate a loss-of-function allele in the mouse in order to examine whether ADAMTS-16 functions in mouse testis determination or testicular function. Male mice lacking Adamts16 on the C57BL/6N background undergo normal testis determination in the fetal period. However, adult homozygotes have an average testis weight that is around 10% lower than age-matched controls. Cohorts of mutant males tested at 3-months and 6-months of age were fertile. We conclude that ADAMTS-16 is not required for testis determination or male fertility in mice. We discuss these phenotypic data and their significance for our understanding of ADAMTS-16 function.

Identifying and addressing training deficiencies in the examination of cryptorchidism—a quality improvement study

Accurate and timely diagnosis of cryptorchidism by primary care providers (PCPs) is critical to oncologic and fertility outcomes. Physical exam is the mainstay of diagnosis, but little is known about PCPs' skills in examining cryptorchid patients. Patients referred to surgeons for cryptorchidism often have normal or retractile testes on exam, and delayed or missed diagnosis of cryptorchidism may contribute to advanced age at surgical intervention. Previous studies on cryptorchidism have not investigated the baseline training, confidence, and/or exam skills of providers. The authors aimed to define baseline training and provider confidence in the exam of cryptorchid patients and to improve examiner confidence using bedside teaching with a pediatric urologist. Secondarily, baseline training and confidence were correlated to skill. Medical students, family medicine, pediatrics, and urology residents, and pediatric attendings completed surveys on baseline training and self-reported confidence in the examination of cryptorchid patients at an academic institution from 2017 to 2018. N.G.K. (pediatric urologist) proctored examinations of cryptorchid patients and provided standardized grades and individualized feedback. Surveys were readministered after 3 months. Non-parametric comparison tests were performed to determine intervention effect and compare subgroups. Ninety-two respondents participated. 62% reported little to no formal training on the scrotal exam, 50% were self-taught, and 20% defined undescended testis incorrectly. Confidence increased with level of training, comparing attendings to residents to students (P <0.001). Those who learned from a mentor had higher baseline confidence than those who did not (P<0.01). Baseline confidence and amount of formal training positively correlated with exam skill as graded during proctored sessions (n=59, P<0.01). Provider confidence was higher after proctored exams (Fig. 2, n = 32, P<0.0001). Significant training deficiencies exist in the examinationof cryptorchid patients. A single proctored exam with a pediatric urologist can improve provider confidence and may improve exam skills. A rotation with pediatric urology, including proctored exams of cryptorchid patients, has become standard practice for pediatric trainees at the authors institution as a result of this study. While further studies are required to assess the effectiveness of bedside teaching and its impact on accurate and timely diagnosis of cryptorchidism, implementation of the authors quality improvement recommendations at other teaching institutions would help address training deficiencies in the examination of cryptorchid patients.

Treatment of Testicular Relapse of B-cell Acute Lymphoblastic Leukemia With CD19-specific Chimeric Antigen Receptor T Cells

BackgroundIrradiation has been a standard treatment for testicular relapse but is associated with severe hypogonadism. Because CD19-specific chimeric antigen receptor T (CAR-T) cells can eradicate leukemic blasts in cerebrospinal fluid, a pharmacologic sanctuary site, we tested the efficacy of this therapy in 7 boys with isolated testicular relapse of B-cell acute lymphoblastic leukemia. Patients and MethodsCD19-specific CAR-T cells were generated with the use of autologous T cells transduced with a lentiviral vector to express a CAR molecule containing anti-CD19 scFv derived from the HI19α murine monoclonal antibody, human CD8α hinge, and human 4-1BB (CD137) and CD3ζ costimulatory signaling transmembrane domains. After the conditioning regimen, which consisted of intravenous fludarabine and intravenous cyclophosphamide, 7 patients with a median age of 9 years (range, 2-10 years) with isolated testicular relapse received a single infusion of CD19 CAR-T cells at a total dose of 5 × 106 all T cells per kilogram. ResultsAll 7 patients achieved complete remission with normal testes. Six patients remained in second remission for 5 to 23 months (median, 14 months), and 1 patient subsequently relapsed in the bone marrow. The probability of event-free survival for all patients at 12 months of follow-up was 83.3% ± 15.2% (standard error). The treatment was well-tolerated, with grade 1 cytokine-release syndrome developing in 5 patients. ConclusionThese results suggest that CAR-T cell therapy is a treatment option for patients with testicular relapse.

Revisiting the success rate of one-stage Fowler-Stephens orchiopexy with postoperative Doppler ultrasound and long-term follow-up: a 15-year single-surgeon experience

The optimal management of a high undescended testicle (UDT) remains debated. When tethering is due to shortened spermatic vessels, a one-stage or two-stage Fowler-Stephens Orchiopexy (FSO) can be performed. Published series suggest a higher success rate with a two-stage FSO, though its superiority has not been ascertained. The authors examine patient and surgical factors associated with success of one-stage FSO and compare our outcomes with the established literature. We retrospectively reviewed the charts of 43 boys who underwent one-stage FSO for 45 testes from 2003 to 2018 by a single surgeon at our institution. Patient factors, surgical approach, and postoperative results were reviewed. Radiographic success was a testis with intact vascular flow or normal echotexture on scrotal ultrasound. Clinical success was a non-atrophic, intrascrotal testis at postoperative follow-up. Radiographic and clinical success was compared. Data analysis was performed with Fisher's exact test and t-test for categorical and continuous variables respectively. Follow-up (mean 29 months; 0.5 months-14.5 years) was available for 40 patients (42 testes; 21 bilateral, 21 unilateral). Average age at surgery was 21.8 months (4 months-10.4 years). Associated conditions were seen in 14 patients. Twenty-nine patients had postoperative scrotal Doppler ultrasound with radiographic success in 25/29 (86.2%). Overall clinical success was 34/42 (81%) with 4 (9.5%) each of atrophy and ascent. A second surgery to address ascent in four patients allowed correction in three for a success rate of 88%. Radiographic and clinical success was significantly associated (P=0.01). Clinical success was also significantly associated with inguinal rather than laparoscopic testis mobilization (P=0.03) but not to patient's age, associated conditions, unilaterality/bilaterality, or pre-operative hypoplasia or location. Greater than 3 years of follow-up was available in 11 (26.2%) cases, of which seven had successful results. All adverse outcomes occurred within 8 months after surgery. Success was otherwise durable. This is the largest series of one-stage FSO to date. The success rate of one-stage FSO in this series matches that published in the literature for two-stage FSO; equal success is achieved with fewer surgeries. Postoperative Doppler ultrasound demonstrating intact vascular flow or normal testis echotexture is significantly associated with clinical success and highlights the utility of postoperative ultrasound in patients with an equivocal exam.

Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.