Prepubertal Tissue Research Articles

Metabolic transitions define spermatogonial stem cell maturation.

Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

Fertility preservation for prepubertal boys: are we ready for autologous grafting of cryopreserved immature testicular tissue?

Fertility preservation in prepubertal boys is a matter that has been gaining ground fast during the past 20 years. As the life expectancy for childhood cancer survivors continues to increase (5-year survival rate>80%), and as the risk of permanent infertility in adulthood is still high with current treatment options, offering a testicular biopsy to cryopreserve immature testicular tissue (ITT) containing spermatogonial stem cells is now recommended as an experimental option for fertility preservation.Transplantation of cryopreserved testicular prepubertal tissue fragments back to the cured patient appears to be one of the most feasible and promising techniques for fertility restoration. In 2019, the birth of a healthy female baby rhesus monkey after intracytoplasmic sperm injection using spermatozoa recovered from frozen-thawed ITT autografts fueled the hopes of translating the technique to humans.While gonadal tissue autotransplantation has already been successfully achieved in clinical practice for females, it has never been attempted in males to date.In this work, we review acquired knowledge on ITT transplantation in animal models and share our vision on the remaining challenges to autograft currently banked human ITT within pilot clinical trials in the near future.

P–056 To graft or not to graft? Intratesticular grafting of testicular tissue from Klinefelter boys to the mouse testis as possible novel in vivo model

Abstract Study question Can intratesticular transplanted testis tissue from Klinefelter boys to the mouse testis be used to study the mechanisms behind testicular fibrosis? Summary answer Grafting of testicular tissue from Klinefelter boys to the mouse testis is not a valuable new in vivo model to study Klinefelter-related testicular fibrosis. What is known already Klinefelter syndrome (KS; 47, XXY) affects 1–2 in 1000 males. Most KS men suffer from azoospermia due to a loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. An optimal in vivo model to study the KS testicular fibrotic process is not available. This study aimed to evaluate a possible in vivo model to study KS-related testicular fibrosis. In addition, the effect of the mast cell blocker ketotifen, which showed positive effects on fertility in infertile non-KS patients, was evaluated in this graft model. Study design, size, duration First, the survival time of the KS graft was established, since it was the first time KS tissue was transplanted to the mouse testis. Testes were collected after two, four, six and eight weeks after which histological and immunohistochemical evaluations were performed. Next, the effect of daily ketotifen injections on the fibrotic appearance of intratesticular grafted testicular tissue from KS and controls was evaluated. Participants/materials, setting, methods Testicular biopsy samples from pre- and peripubertal KS (n = 22) and age-matched control samples (n = 22) were transplanted to the testes of six weeks old Swiss Nu/Nu mice (n = 22). Prior to grafting, testicular tissue pieces were cultured in vascular endothelial growth factor (VEGF) for five days. Next, tissues were transplanted to the mouse testes. Testicular transplants were analysed by immunohistochemistry. In the second experiment, mice were given daily subcutaneous injections of ketotifen or saline. Main results and the role of chance Four weeks after transplantation, all KS grafts could still be retrieved. At a later timepoint, degeneration of the tissue could be detected. In the grafts, recovered four weeks after transplantation, about 30% of the tubules in peripubertal grafts showed a good integrity, while in the prepubertal tissue, 83% of the tubules were intact. A fibrotic score was assigned to each graft. No significant changes in fibrotic score was observed between testicular biopsies before or after transplantation. However, an increased (p &amp;lt; 0.01) fibrotic score was observed after in-vitro treatment with VEGF both in control and KS tissue. Based on recovery and tubule integrity grafts were recovered after four weeks in the second experiment. Treatment with ketotifen did not result in significant histological differences compared to non-treated grafts (KS and control tissue). The survival potential of grafts from KS testicular biopsies of pre- and peripubertal boys was patient- and age-dependent. After four weeks, most KS tissue starts to degenerate. In prepubertal tissue, seminiferous tubules were mostly intact, while tissue from adolescent boys was impaired. Interestingly, no loss of germ cells was observed after transplantation of the testicular tissue. Limitations, reasons for caution The availability of tissue from young KS patients is very scarce, leading to a low number of included patients (n = 8). Testicular tissue pieces from the same patient were included to evaluate the differences before and after transplantation. However, histological variability between testicular tissue biopsy pieces is well-known in KS patients. Wider implications of the findings Since testicular tissue from KS boys, transplanted to the mouse testes, already starts to degenerate after four weeks and the integrity is not optimal, we conclude that this is not a valuable model for future studies. In vitro models to study the KS-testicular fibrosis should be investigated. Trial registration number NA

P–699 Multi-scale study of the architecture, topography and mechanics of the human ovary from prepuberty to menopause: a blueprint for next-generation bioengineering and diagnosis

Abstract Study question Does the ovarian ECM have a precise and unique biophysical phenotype, specific to each age, from prepuberty to menopause? Summary answer Differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. What is known already Ovarian engineering has recently emerged to respond to patient needs and offer reliable models for basic research. It has relied on synthetic and natural biomaterials and microfluidics. However, these techniques were designed based on knowledge acquired from 2D cell culture and animal models. Our lack of information on the human ovary hampers our ability to mimic the main features of this organ, for clinical applications. The complex composition and hierarchical structure of its ECM complicates the design of truly biomimetic constructs, notably: fiber morphology, interstitial and perifollicular fiber orientation, porosity, topography, and viscoelasticity, which all play a role in mechanotransduction. Study design, size, duration Ovarian biopsies were taken from prepubertal (mean age [±SD]=7±3 years, n = 21), reproductive age (mean age [±SD]=27±5, n = 26 ) and menopausal (mean age [±SD]=61±6 years, n = 29) patients after obtaining their informed consent. All participating adult subjects were undergoing laparoscopic surgery for benign gynecological diseases not affecting the ovaries. Prepubertal tissue was derived from young cancer patients scheduled for ovarian cortex cryopreservation as a fertility preservation strategy, before being subjected to acute gonadotoxic cancer treatments. Participants/materials, setting, methods All samples were cryopreserved by slow freezing and kept frozen until the day of their analysis. Tissues provided from the same patients (n = 5 per age group) were investigated by scanning electron microscopy (SEM) (fiber, pore and topography analyses) and atomic force microscopy (AFM). A larger number of paraffin-fixed biopsies (prepubertal, n = 16, reproductive-age, n = 21, and menopausal, n = 24) obtained from the biobank of St-Luc’s Hospital were used to conduct computed fiber orientation analysis. Main results and the role of chance Our results revealed a unique ECM architecture at reproductive age, where fibers of intermediate diameter are assembled into thickest bundles compared to prepubertal and menopausal tissues(p &amp;lt; 0.0001). Indeed, during prepuberty the bundles assemble into a tight network with high number of small pores while reproductive-age ovary gain more porosity(p &amp;lt; 0.0001). However, at menopause tissue pore number and area change significantly(p &amp;lt; 0.001). These pore geometry and distribution changes contribute to diffusion and access of key molecules to/from cells, which can be translated into changes in permeability and molecule selectivity with age. Fiber directionality around follicle borders at preantral stages revealed that before and after puberty, secondary follicles appear to modify their microenvironment arrangement locally compared to follicles at earlier stages of development (p &amp;lt; 0.01), by reorienting the majority of collagen fibers below 50°.This could indicate that follicles at this stage require higher fiber contact and adhesion signaling to complete their development and maturation towards ovulation. AFM evidenced a relatively rigid ovarian tissue at prepuberty, softening significantly at reproductive age, then stiffening considerably upon menopause. These differences(p &amp;lt; 0.01) are not only structure-dependent, but also related to biochemical differences in ECM composition, as previously demonstrated in our follow-up of variations in elastic matrisome components from prepuberty to menopause. Limitations, reasons for caution The samples represent single time points from each age group which could present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. Wider implications of the findings: Our study provides the first conclusive proof of a link between ECM biophysics and fertility by comparing different stages of ovarian transformation related to a woman’s reproductive life, which will oriente new strategies for infertility prognoses based on ECM biophysics and may become a blueprint for designing functional engineered ovaries. Trial registration number Not applicable

O-190 Comparison between effects of exposure to platinum-based chemotherapeutics (cisplatin and carboplatin) on Sertoli cell number and functions in immature human testicular tissues

Abstract Study question Does exposure to either cisplatin or carboplatin have a damaging effect on the Sertoli cell population in the immature human testicular tissues? Summary answer Exposure to cisplatin or carboplatin did not appear to have a major effect on Sertoli cell number or function in the immature human testicular tissues What is known already Long-term survival rates for children with cancer are more than 80%. However, childhood cancer treatment may result in subsequent infertility. Cisplatin is one of the most commonly used drugs for childhood cancers. Carboplatin, a second generation platinum drug, is administered at 10-times the dose of cisplatin and is believed to be less gonadotoxic. In our recent publication we have shown that exposure to both cisplatin and carboplatin acutely reduce the germ cell number in immature human testicular tissues. However, it is not known how cisplatin and carboplatin affect Sertoli cell number and function. Study design, size, duration In-vitro culture of human fetal and pre-pubertal testicular tissues was utilised. Tissue pieces were cultured for 1-3 days prior to exposure to clinically-relevant doses of chemotherapeutics or vehicle control for 24hrs in two sets of experiments: 1) 0.5 or 1 μg/ml cisplatin and culture ended at 24 and 96hrs post-exposure (fetal only); 2) 0.5 μg/ml cisplatin or 5 ­μg/ml carboplatin until 72 (both fetal and pre-pubertal) and 240hrs post-­exposure (fetal only). Participants/materials, setting, methods Testicular tissue fragments from second trimester human fetal (14-22 gestational weeks; n = 3-6) or pre-pubertal patients (1-8years old; n = 5) were cultured in a ‘hanging drop’ system.Quantification of Sertoli cell number (cells per cord/tubular area (mm2)) was performed on sections stained for expression of SOX9. Culture medium was collected to measure levels (ng/ml) of Anti-Mullerian hormone (AMH) and Inhibin B using ELISA. Statistical analysis was performed using two-way ANOVA to account for inter-individual variation between fetuses/patients. Main results and the role of chance Quantification of positively stained Sertoli cells showed that exposure to both doses of cisplatin had no effect on Sertoli cell number at 24 and 96hrs post-exposure. No changes in AMH and inhibin B levels were observed at these time-points. Comparison between cisplatin- or carboplatin-exposed human fetal testicular tissues showed no difference in Sertoli cell numbers at either 72hrs or 240hrs post-exposure. No difference in Sertoli cell number was observed in pre-pubertal testicular tissues exposed to either cisplatin or carboplatin at 72hrs post-exposure. Limitations, reasons for caution Human fetal and pre-pubertal testis tissue is of limited availability, thus, sample sizes used in this study were relatively low. ‘Hanging drop’ culture might not recapitulate all in-vivo aspects of immature testis microenvironment. Wider implications of the findings Exposure to cisplatin or carboplatin did not affect Sertoli cell number in the immature human testicular tissues. Taken together with our recent publication, this suggests that these two platinum-based chemotherapeutic agents cause direct damage to germ cells. Functionality of Sertoli cells in chemotherapy-exposed tissues need to be further investigated. Trial registration number not applicable

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

A prospective study on the long-term outcome of prepubertal and pubertal boys undergoing testicular biopsy for fertility preservation prior to hematologic stem cell transplantation.

Few studies have reported the long-term outcomes of prepubertal and pubertal boys undergoing testicular biopsy for fertility preservation (FP). This prospective longitudinal study examined 21 boys (aged 1.5-14.5 years) who underwent testicular biopsy for FP prior to allogeneic (n=20) or autologous (n=1) hematological stem cell transplantation (HSCT) between 2003 and 2010. During counseling, pubertal boys were encouraged to produce a sperm sample by masturbation , while prepubertal boys were presented with surgical testicular tissue retrieval as an option for experimental FP. Clinical outcomes included postoperative complications, pubertal development, and sex-hormone levels. Survivors approaching adulthood were encouraged to provide semen samples. Twenty boys, including 14 in prepuberty and six in early puberty (Tanner stage 2-3), underwent open testicular biopsies. Two pubertal biopsies contained mature sperms, which were cryopreserved. Testicular tissue was vitrified in the remaining 18 cases. One pubertal boy (Tanner stage 4) underwent percutaneous testicular sperm aspiration and sperms obtained were cryopreserved. Postoperative complications (hematoma or infection) were rare. Overall, 14 boys survived>5 years (mean follow-up after HSCT, 7.2 years) and 11 showed advanced puberty. Semen samples were provided by five boys and obtained sperm were cryopreserved from two. Individuals at adulthood had normal testosterone levels but subnormal testicular size, high follicle stimulating hormone, and low inhibin B and anti-Müllerian hormone levels. No long-term risks were detected during continuous clinical follow-up. Experimental testicular biopsies for FP were well accepted by the patients and families, despite the absence of methods to use prepubertal tissue for fertility treatment.

Vaginal Discharge

Key Points Vaginal discharge is caused by infection, inflammation, or changes in the vaginal flora. Vaginal discharge in the prepubertal child can be caused by infection, congenital abnormalities, trauma, or dermatologic conditions. Nonspecific vaginitis is the most common gynecologic problem in the prepubertal child. Prepubertal hypoestrogenic tissues are more susceptible to local irritants, foreign bodies, and infections. Most common causes of vaginitis in postpubertal women are bacterial vaginosis and vaginal candidiasis (Candida vulvovaginitis). Laboratory documentation of etiology of vaginal discharge is mandatory prior to treatment. Target treatment to etiology.

Immunohistochemical characterization of the anti-M\xfcllerian hormone receptor type 2 (AMHR-2) in human testes

PurposeIn males, AMH is secreted by immature Sertoli cells; following exposure to endogenous androgens, Sertoli cells undergo a process of maturation which ultimately inhibits AMH expression to undetectable levels in the serum. However, expression of AMH receptor (AMHR-2) has never been studied in human testes, and high intratubular concentrations of AMH have been reported in recent literature. We therefore assessed expression of AMHR-2 in several testicular tissue samples by immunohistochemistry (IHC).MethodsThe IHC method was first validated on tissue samples from healthy human testis (n = 2) and from marmoset ovary (n = 1). The same method was then used for assessment on testicular histopathology specimens from patients with mixed atrophy (MA, n = 2), spermatogenetic arrest (SA, n = 2), Sertoli cell-only syndrome (SCO, n = 1), Klinefelter syndrome (KS, n = 1), and nonseminomatous germ cell tumors (NSGCT, n = 1). Tissue samples from two subjects at different pubertal stages (AndroProtect (AP), aged 5 and 14 years) with hematological malignancies were also retrieved.ResultsIn adult men, AMHR-2 was expressed on peritubular mesenchymal cells, with patterns closely mirroring α-smooth muscle actin expression. Similar patterns were preserved in almost all conditions; however, in nonseminomatous germ cell tumors the tissue architecture was lost, including AMHR-2 expression. More positive and diffuse staining was observed in tissue samples from prepubertal testes.ConclusionsIn specimens from both healthy and affected testes, AMHR-2 expression appears weaker in adult than in prepubertal tissue sections. The persistence of AMHR-2 expression seemingly hints at a possible effect of intratesticular AMH on the tubular walls.

Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds

In vitro spermatogenesis (IVS) has already been successfully achieved in rodents by organotypic and soft matrix culture systems. However, the former does not allow single cell input, and the latter presents as a simple thick layer in which all cells are embedded. We explored a new culture system using a mouse model by employing an alginate-based hydrogel and 3D bioprinting, to control scaffold design and cell deposition. We produced testicular constructs consisting of printed cell-free scaffolds (CFS) with prepubertal testicular cells (TC) in their easy-to-access macropores. Here, the pores represented the only cell compartment (TC/CFS). Double-cell compartment testicular constructs were achieved by culturing magnetic-activated cell sorting-enriched epithelial cells in the pores of interstitial cell-laden scaffolds (CD49f+/CLS). Cell spheres formed in the pores in the weeks following cell seeding on both CFS and CLS. Although restoration of the tubular architecture was not observed, patches of post-meiotic cells including elongated spermatids were found in 66% of TC/CFS. Differentiation up to the level of round spermatids and elongated spermatids was observed in all and 33% of CD49f+/CLS constructs, respectively. Organ culture served as the reference method for IVS, with complete spermatogenesis identified in 80% of cultivated prepubertal tissue fragments. So far, this is the first report applying a 3D bioprinting approach for IVS. Further optimization of the scaffold design and seeding parameters might be permissive for tubular architecture recreation and thereby increase the efficiency of IVS in printed testicular constructs. While it remains to be tested whether the gametes generated on the alginate-based scaffolds can support embryogenesis following IVF, this IVS approach might be useful for (patho)physiological studies and drug-screening applications.

In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. N/A. Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

Is the protein expression window during testicular development affected in patients at risk for stem cell loss?

Is the protein expression window during testicular development affected in prepubertal patients at risk for stem cell loss? Nuclear ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression in Sertoli cells and interstitial expression of inhibin α (INHA), sex-determining region Y-box 9 (SOX9) and steroidogenic acute regulatory protein (STAR) was affected in patients with Klinefelter syndrome. Some patients undergoing testicular tissue banking have already been treated before the testis biopsy is taken. These treatments include chemotherapy or hydroxyurea, which can have an influence on the stem cell number and function. A germinal loss occurs in Klinefelter patients, but its cause is currently unknown. Parrafin-embedded testicular tissue from 5 fetuses, 25 prepubertal patients and 5 adults was used to characterize the spatial and temporal distribution of different testicular marker proteins during testicular development. Expression of the markers was evaluated in germ cells, Sertoli cell and interstitial cells. The integrity of this time window was analyzed in patients at risk for germ cell loss: patients treated with hydroxyurea (n = 7), patients treated with chemotherapy (n = 6) and patients affected by Klinefelter syndrome (n = 5). Immunohistochemistry was performed in normal fetal, prepubertal and adult testicular tissue to set up a timeline for the expression of melanoma antigen family A4 (MAGE-A4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4), homeobox protein NANOG, INHA, anti-Müllerian hormone, androgen receptor (AR), SOX9 and STAR. The established timeline was used to evaluate whether the expression of these markers was altered in patients at risk for germ cell loss (patients treated for sickle cell disease (hydroxyurea) or cancer (chemotherapy) and patients with Klinefelter syndrome). A protein expression timeline was created using different markers expressed in different testicular cell types. Less positive tubules and less positive cells per tubule were observed for MAGE-A4 and UCHL1 expression in the KS compared with the non-treated group (P < 0.01). Higher nuclear UCHL1 Sertoli cell expression was observed in the KS group compared with the non-treated group (P < 0.05). Higher interstitial expression of INHA (P < 0.05), SOX9 (P < 0.01) and STAR (P < 0.05) was observed in KS compared with the non-treated group. Important age variations exist in the prepubertal groups. Therefore, data were represented in three age groups. However, owing to the limited access to prepubertal tissue, no statistical comparison was possible between these groups. For the Klinefelter group, tissue was only available from patients older than 12 years. The expression timeline can add knowledge to the process of spermatogenesis and be used to evaluate altered protein patterns in patients undergoing potentially gonadotoxic treatments, to monitor spermatogenesis established in vitro and to unravel causes of germ cell loss in Klinefelter patients.

What is the best cryopreservation protocol for human testicular tissue banking?

Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking? Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue. Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue. Fragments (n = 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (-S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV). Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types. The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 ± 5.6 in control tissue to 4.9 ± 2.1, 8.2 ± 5.4, 11.6 ± 5.1, 8.8 ± 3.9, 12.6 ± 4.4 and 11.7 ± 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO -S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P < 0.001). Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue. An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.

Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant

To study the effect of age and sample state on cryopreservation of testicular tissue, evaluate toxicity of commonly used cryoprotectants (CPs), and determine their optimal concentration for use. Prospective experimental study. Academic research unit. Patients with prostate carcinoma undergoing orchidectomy. We also studied immature and adult male Holtzman rats. Toxicity of CPs before freezing, morphology, and relative viability after freezing were evaluated for rat testicular cell suspensions (CS) and tubular fragments (TUB). Relative viability of adult human testicular CS and TUB after thaw was evaluated. Human TUB were cultured after thaw for 48 hours in medium containing epidermal growth factor (EGF), and effects on viability, morphology, and gene expression were determined. Viability and ploidy were measured with flow cytometry, postthaw cryodamage of immature rat tissue was studied by transmission electron microscopy, cell proliferation and differentiation were evaluated by immunohistochemistry and by real-time polymerase chain reaction. Immature testicular tissue was more susceptible to toxic assault by CP than adult tissue and displayed cell-specific sensitivity to CP, with glycerol, dimethyl sulfoxide and ethylene glycol being effective in protecting spermatid (1N), spermatogonia (2N) and spermatocyte (4N) populations respectively. Preservation as TUB may be preferred over CS and DMSO is an effective CP for immature and ethylene glycol for adult testicular tissue. Differential sensitivity of immature testicular tissue to CPs warrants judicious selection of CP on the basis of end application for prepubertal tissue.

Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant

Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved-thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2 ± 4.5% (fresh) to 13.6 ± 1.8 (DMSO), 9.5 ± 1.7 (PrOH), or 6.8 ± 1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2 ± 2.5%) and primary follicles (28.1 ± 5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2 ± 3 and 5.4 ± 2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.

Immunocytochemical localization of angiotensin II receptor subtypes 1 and 2 in the porcine fetal, prepubertal and postpubertal ovary.

There is considerable evidence for a mammalian ovarian renin-angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors--type 1, AT1 and type 2, AT2. We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT1 and AT2 receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two-cell layer at the ovarian periphery. In prepubertal tissue, positive AT1 and AT2 staining was localized to granulosa cells adjacent to the basement membrane of pre-antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT1 and AT2 immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT1 staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT1 receptors than AT2 receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development.

Autoradiographic determination of angiotensin II receptors in prepubertal and postpubertal pig ovarian tissue

The aim of this study was to determine whether specific receptors for angiotensin II are present in prepubertal and postpubertal pig ovaries using an autoradiographic technique and computerized densitometry. Frozen sections were cut from prepared samples, and (125)I-labelled angiotensin II and the angiotensin II receptor subtype-specific nonpeptide antagonists for receptor subtype 1, AT(1) (GR117289) and subtype 2, AT(2) (PD123319) were used. In both pre- and postpubertal pig ovarian tissue, specific receptors for angiotensin II were demonstrated. These receptors had a density of 2487.6 (range: 267.5-5177.6, n = 4) and 3703.8 (range: 1819.9-5207.8, n = 4) fmol per mm(2), respectively, and dissociation constants of 130.0 and 26.3 nmol l(-1), respectively (prepubertal ovarian range: 106.0-165.4 nmol l(-1); postpubertal ovarian range: 26.1-100.3 nmol l(-1); P < 0.05, Mann-Whitney U test). AT(1) receptors with a K(i) for (125)I-labelled angiotensin II of 346.9 nmol l(-1) in the prepubertal and 268.1 nmol l(-1) in the postpubertal ovary were located predominantly in follicle wall tissue. Competitive inhibition studies using both angiotensin II antagonists resulted in a decrease in K(i) with prepubertal tissue (283.7 nmol l(-1)) and an increase in postpubertal tissue (293.9 nmol l(-1)). Immunocytochemistry using sections from paraffin wax-embedded prepubertal (n = 4) and postpubertal (n = 4) pig ovaries confirmed the presence of AT(1) receptors on the granulosa cell layer, but not the thecal cell layer, of antral follicles in both pre- and postpubertal pig ovarian tissue, and AT(2) receptors within the granulosa cell layer of prepubertal pig ovarian antral follicles. In summary, these results indicate that angiotensin II receptors are of higher affinity in postpubertal tissue than they are in prepubertal tissue, and indicate an active renin-angiotensin system within the pig ovary.

Sperm and ova conservation: existing standards of practice in North America.

Rapid advances have occurred in both reproductive medicine and survival from childhood cancer. To establish the current level of best clinical practice for sperm, ovarian, and prepubertal tissue collection and storage, a cross-sectional survey of a major pediatric oncology collaborative study group (Pediatric Oncology Group, POG) was performed. Of the 110 centers surveyed, 69 questionnaires (63%) were completed. No responding center had guidelines regarding which young people should be offered sperm, ovarian, or prepubertal testicular tissue conservation; 93% centers had offered sperm and 10% ova conservation; 15% had offered sperm conservation to males prior to completion of sexual development and 3% to girls prior to sexual maturation. All centers were more likely to offer sperm conservation than ova conservation for any given disease. The most common diseases for which conservation was offered were Hodgkin and non-Hodgkin lymphoma, and sarcomas. Fertility counseling was offered in a variety of settings by 71% of centers by health care professionals, including doctors, nurses, social workers, psychologists, and geneticists. There was little agreement regarding appropriate indications for, and method of, gamete preservation in children's cancer centers. It is hard to establish best clinical practice from these data. Unresolved medical, legal, and ethical issues necessitate the development of a voluntary code of practice and guidelines in order to ensure good clinical practice.

Effect of pubertal development on estrogen receptor levels and stromal morphology in the guinea pig prostate.

Using an immunocytochemical assay (ERICA) with a monoclonal antibody (H222Sp gamma) to the human estrogen receptor, we have demonstrated a stromal localization of the estrogen receptor in the dorsolateral prostate of the guinea pig. Specific staining of estrogen receptor in the guinea pig prostate was confined to the nuclei of periacinar and interacinar stromal cells. In comparison with prepubertal tissues, estrogen receptor staining intensity was markedly reduced in postpubertal prostatic tissues. No immunoreactive estrogen receptor was detected in the acinar epithelial cells irrespective of the developmental stage of the guinea pig prostate. Electron microscopic examination of the guinea pig prostate showed that the stromal component consists predominantly of smooth muscle cells, which, during pubertal development, undergo marked cytological changes and increase in size. These changes in the prostatic stroma were associated with a greater than fivefold reduction in levels of cytosolic and nuclear estrogen receptor determined by either a radioligand binding assay or an enzyme immunoassay (EREIA) and expressed relative to soluble protein. Morphometric analysis of the prostatic stromal cell density (SCD: nuclei/mm2 interacinar stroma), which is inversely proportional to stromal cell size, indicated that the SCD decreased approximately threefold during pubertal development. Furthermore, cytosolic estrogen receptor levels in mechanically separated prostatic stromal fractions were found to vary concordantly with the SCD during pubertal development. To determine whether estrogen influences normal development of the guinea pig prostate, the effect of various hormonal manipulations on stromal development was examined. Castration of prepubertal animals prevented the threefold decrease in SCD that is characteristic of pubertal development. Treatment of prepubertal castrates with estradiol and 5 alpha-dihydrotestosterone (DHT) in combination over a period equivalent to the transpubertal growth phase resulted in a stromal cell density similar to that seen in prostatic sections from intact postpubertal animals. In contrast, treatment of prepubertal castrates with either estradiol or DHT alone resulted in a prostatic stromal cell density intermediate between that observed in intact prepubertal and postpubertal animals. These findings suggest that both estrogen and androgen are required for the normal development of the guinea pig prostatic stroma.