Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Sperm chromatin condensation defects, but neither DNA fragmentation nor aneuploidy, are an independent predictor of clinical pregnancy after intracytoplasmic sperm injection.

PURPOSE: The impact of sperm DNA damage on intracytoplasmic sperm injection (ICSI) outcomes remains controversial. The purpose of the study was to evaluate the prognostic value of several types of sperm nuclear damage on ICSI clinical pregnancy. METHODS: Our retrospective study included a total of 132 couples who consulted for male or mixed-factor infertility that benefited from ICSI cycles from January 2006 to December 2015. All infertile males presented at least one conventional semen parameter alteration. Sperm nuclear damage was assessed using the Motile Sperm Organelle Morphological Examination for sperm head relative vacuolar area (RVA), aniline blue staining for chromatin condensation, terminal deoxynucleotidyl transferase dUTP nick-end labeling for DNA fragmentation, and fluorescence in situ hybridization for aneuploidy. RESULTS: Infertile males who achieved pregnancy after ICSI had fewer chromatin condensation defects than did males who did not achieve any pregnancy (15.8 ± 12.0% vs. 11.4 ± 7.9%, respectively, P = 0.0242), which remained significant in multivariate regression analysis (RR = 0.40 [0.18 to 0.86], P = 0.02). RVA, DNA fragmentation, and aneuploidy were not predictive factors of ICSI outcomes. The pregnancy rate was significantly decreased by number of progressive motile spermatozoa with normal morphology after migration (P = 0.04). In female partners, 17ß estradiol of less than 2000 pg/mL on the day of ovulation induction significantly reduced the occurrence of clinical pregnancy (P = 0.04). CONCLUSION: Sperm chromatin condensation defects were more frequently observed in couples with ICSI failure and should be considered a negative predictive factor for the occurrence of clinical pregnancy.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

STUDY QUESTION: Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? SUMMARY ANSWER: The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. WHAT IS KNOWN ALREADY: Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. STUDY DESIGN, SIZE, DURATION: Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. MAIN RESULTS AND THE ROLE OF CHANCE: Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Twenty-four-month oral carcinogenicity study of ebrotidine in rats.

Three groups of Sprague-Dawley CD rats (males and females) were initially administered p.o. with ebrotidine, a novel H2-receptor antagonist, mixed with the diet, at 50, 200, and 500 mg/kg/d, respectively. Two concurrent control groups of animals were used. After 13 months, initial 200 mg/kg was lowered to 150 mg/kg, and a new group was administered with 300 mg/kg, due to the body weight reduction observed in the top dose group. After 24 months, survivors were killed and necropsied, and a histopathological study was performed. The frequencies of the different tumour types that were found were not raised due to the treatment. Lower frequencies of some types of pituitary and mammary gland tumours, in the groups treated with the higher doses, were the only statistically significant changes. Among the non-neoplastic effects, a lower body weight increment and food consumption (500 and 300 mg/kg, both sexes), lower survival (500 mg/kg, males), presence of lipoid pneumonia (500 mg/kg, only in males, and 300 mg/kg, both sexes), and lithiasis in urinary system (500 mg/kg) were observed. No changes in gastric mucosa (the main target organ) were attributable to ebrotidine. Regarding the non-neoplastic effects, 150 mg/kg was the no observed adverse effect level. According to the previous results of the carcinogenicity study in mice, conjointly with those of the study in rats reported here, there is no evidence of carcinogenic risk either in males or in females in these species.

Carcinogenicity studies on ebrotidine.

The results from two carcinogenicity studies on ebrotidine (N-[2-(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) conducted in mice and rats are reported. Oral doses of 50, 200 and 500 mg/kg were administered to mice for 18 months and 50, 200 (150), 300 and 500 mg/kg were administered to rats for 24 months. The study design was prepared according to EEC guidelines, and the recommendations by the International Agency for Research on Cancer were used for the statistical analysis of data. Weekly palpations were made along the course of studies and general parameters were monitored. The only effects attributed to ebrotidine administration were a slight decrease in the survival rate of female mice given the 500 mg/kg dose and a lower weight gain in rats of both sexes. The histopathological data revealed that lipoid pneumonia and kidney calculi are more frequent in rats treated with doses of 500 and 300 mg/kg. No increment in the spontaneous occurrence of tumours or significant presence of tumours in treated animals differing from that in control animals was observed, and a decrease in the time required for their onset that could be related to ebrotidine was not observed either. There were no differences in hyperplastic and/or dysplastic changes between treated and control animals. Therefore, it is deduced that ebrotidine does not induce neoplastic or preneoplastic effects in rats or mice even at doses of 500 mg/kg, at which some general toxicity effects are seen.

[A single bone metastasis as the initial manifestation of an undifferentiated bladder carcinoma]. / Metástasis ósea única como manifestación inicial de un carcinoma indiferenciado de vejiga.

The spread of Vesical Neoplasia occurs frequently through local or regional extension. Its systemic dissemination happens frequently in advanced phases but it is exceptional as initial manifestation. The case of an undifferentiated vesical carcinoma is described, its presentation was as a big size osteolytic lesion on the sacrum. Local manifestations of the tumor appeared later. The study performed both in bones as well as in the vesical mucosa showed an undifferentiated adenocarcinoma. The great resemblance and histological and immunohistochemical concordances of both biopsies confirmed that it was a primary vesical neoplasia with a metastasis in sacrum. The specific clinical evolution of this tumor is discussed which is probably related with the fact that it is an undifferentiated carcinoma.

Adult type of leukodystrophy. Krabbe's disease?

A 24-year-old man developed progressive dementia in seven years. The patient suffered a severe bronchopneumonia and eventually died few days later. Brain coronal sections showed a soft gray-brownish discoloration of white matter of centrum ovale but the subcortical arcuate fibers and the interne capsule were preserved. Microscopically, the white matter showed marked loss of myelin and oligodendrocytes, abundant hypertrophic astrocytes and numerous "globoid cells". The latter showed strong positivity in immunostains for a mouse monoclonal antigalactocerebroside antibody. The presence of these cells in the brain white matter might be the morphological basis to classify the present case as one of Krabbe's Leukodystrophy.

Comparative study of aminoglycoside nephrotoxicity in normal rats and rats with experimental cirrhosis.

Several authors have suggested that the risk of developing aminoglycoside nephrotoxicity is greater in cirrhotic patients than in the noncirrhotic population. However, this has not been confirmed by other investigators. To compare the intensity and characteristics of aminoglycoside nephrotoxicity in cirrhotic and normal rats, 31 rats with carbon tetrachloride-induced cirrhosis with ascites and 35 control rats were treated with gentamicin. Each group of rats was divided into two subgroups in order to receive 10 or 40 mg per kg per day of gentamicin, and different subsets of animals were killed on Days 4, 8 and 12 of treatment for renal histological examination and determination of renal tissue gentamicin concentration. Urine volume, osmolality, sodium excretion and N-acetyl-beta-D-glucosaminidase activity were measured daily throughout the study. Creatinine clearance and trough plasma concentration of gentamicin were determined in each animal immediately before killing. There were no significant differences between cirrhotic and control rats in relation to the magnitude of changes in urine volume, osmolality, sodium excretion and N-acetyl-beta-D-glucosaminidase activity and creatinine clearance during gentamicin administration. The values of a histopathological score semiquantitatively assessing the renal morphological changes observed by light microscopy were not significantly different in cirrhotic and control rats. In addition, similar trough plasma and renal cortical tissue concentrations of gentamicin were observed in both groups of animals. These results suggest that, in this experimental model, cirrhosis does not increase the risk for aminoglycoside nephrotoxicity.

A two-generation reproduction-fertility study of cyanamide in the rat.

A two-generation reproduction-fertility study of cyanamide in the rat has been carried out. After oral administration of 2, 7 and 25 mg/kg/day of the drug, relevant changes have been noted at the highest dose level. Decrease in dam weight gain, in number of corpora lutea, in number of implantations and in number of neonates have been observed in rats of the Fo generation after treatment with 25 mg/kg/day. This group showed a reduced fertility rate and decreases in the weight of several reproductive organs male rats. In contrast to the findings noted in the Fo generation, changes related to cyanamide treatment have not been observed in the F1 generation. Histopathology of these organs has disclosed a low incidence of bilateral testicular atrophy. Decreased fertility rate due to non-specific toxicity associated with a diminished food intake cannot be discarded.

Prevalence of Sjögren's syndrome in autoimmune diseases.

Investigations were carried out in 122 patients in order to identify features of Sjögren's syndrome (keratoconjunctivitis sicca and xerostomia). There were 78 patients with autoimmune diseases (rheumatoid arthritis 21, scleroderma 16, sicca syndrome 16, primary biliary cirrhosis 14, and other autoimmune disorders 11), 11 patients with chronic liver disease other than primary biliary cirrhosis, and 33 patients with a variety of non-autoimmune conditions or no obvious disease. Keratoconjunctivitis sicca was diagnosed by Schirmer's test and rose bengal staining. The oral component was diagnosed by labial biopsy and salivary scintigraphy. Forty nine patients had a definite Sjögren's syndrome, and 77 patients had the syndrome definitely or probably. Definite Sjögren's syndrome occurred in 62% of patients with rheumatoid arthritis, in 69% of patients with scleroderma, and in 71% of patients with primary biliary cirrhosis. Sjögren's syndrome was not present in any of the patients with non-autoimmune conditions. These results show that in an unselected group of patients with Sjögren's syndrome the prevalence of rheumatoid arthritis (26%), scleroderma (22%), sicca syndrome (22%), and primary biliary cirrhosis (20%) is similar. Also the occurrence of Sjögren's syndrome in primary biliary cirrhosis is even higher than that in rheumatoid arthritis.

Main autopsy findings in bone marrow transplant patients.

The autopsy findings in 24 cases (representing 17 males and seven females) that underwent bone marrow transplantation during the course of disease are reported. The mean age of the patients was 20.0 years, with individual ages ranging from 7 to 40 years. In 23 cases, serious infectious disease occurred. A patient frequently exhibited more than one type of infection. In total, there were seven mycotic, eight cytomegalovirus, and 15 bacterial infections. Twenty-three cases showed striking lung pathology, the most prevalent lesions being infectious diseases and those of diffuse alveolar damage, which was found in ten cases. Gastrointestinal tract pathology of differing types was observed in 13 cases. Necrotizing enterocolitis was observed in three of these cases. Three cases also displayed cholangiolar cholestasis, probably related to their terminal infectious disease. In only two cases could graft-vs-host disease be proved histologically.