Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

STUDY QUESTION: Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER: Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY: Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION: We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1-45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION: Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Current recommendations for clinical surveillance and genetic testing in rhabdoid tumor predisposition: a report from the SIOPE Host Genome Working Group.

The rhabdoid tumor (RT) predisposition syndromes 1 and 2 (RTPS1 and 2) are rare genetic conditions rendering young children vulnerable to an increased risk of RT, malignant neoplasms affecting the kidney, miscellaneous soft-part tissues, the liver and the central nervous system (Atypical Teratoid Rhabdoid Tumors, ATRT). Both, RTPS1&2 are due to pathogenic variants (PV) in genes encoding constituents of the BAF chromatin remodeling complex, i.e. SMARCB1 (RTPS1) and SMARCA4 (RTPS2). In contrast to other genetic disorders related to PVs in SMARCB1 and SMARCA4 such as Coffin-Siris Syndrome, RTPS1&2 are characterized by a predominance of truncating PVs, terminating transcription thus explaining a specific cancer risk. The penetrance of RTPS1 early in life is high and associated with a poor survival. However, few unaffected carriers may be encountered. Beyond RT, the tumor spectrum may be larger than initially suspected, and cancer surveillance offered to unaffected carriers (siblings or parents) and long-term survivors of RT is still a matter of discussion. RTPS2 exposes female carriers to an ill-defined risk of small cell carcinoma of the ovaries, hypercalcemic type (SCCOHT), which may appear in prepubertal females. RT surveillance protocols for these rare families have not been established. To address unresolved issues in the care of individuals with RTPS and to propose appropriate surveillance guidelines in childhood, the SIOPe Host Genome working group invited pediatric oncologists and geneticists to contribute to an expert meeting. The current manuscript summarizes conclusions of the panel discussion, including consented statements as well as non-evidence-based proposals for validation in the future.

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.

Skeletal outcome in long-term survivors of childhood high-risk neuroblastoma treated with high-dose therapy and autologous stem cell rescue.

High-dose therapy and hematopoietic stem cell transplantation (HSCT) have been shown to improve survival rates in high-risk neuroblastoma (HR-NBL), but may cause adverse effects on the growing skeleton. We studied skeletal health in a national cohort of long-term survivors of HR-NBL (n=21; age 16-30 years, median 22 years) and in 20 healthy age- and sex-matched controls. In addition to clinical evaluation and measurement of bone mineral density (BMD) by dual-energy X-ray absorptiometry, we performed spinal magnetic resonance imaging. Skeletal complications were categorized according to Common Terminology Criteria for Adverse Events (CTCAE). Altogether, 18/21 survivors presented with at least one skeletal adverse event according to CTCAE, the most common skeletal complications being short stature (n=14) and osteopenia (n=13). Altogether, 38% of the subjects had a severe complication (CTCAE score ⩾3) including bilateral slipped capital femoral epiphyseolysis in 3/21. Fracture rate was not increased. In spinal MRI, no vertebral fractures were found and degenerative intervertebral disc changes were equally prevalent in survivors and controls. BMD was lower in survivors than controls, but differences became non-significant when adjusted for bone size. In conclusion, skeletal late complications are common and can significantly impair the quality of life in young adult survivors of HR-NBL treated with high-dose protocols and HSCT.

Clinical and biological markers of premature aging after autologous SCT in childhood cancer.

The aim of this study was to analyze the prevalence of frailty and physical health limitations among long-term survivors of high-risk neuroblastoma (HR NBL) and to investigate whether frail health is associated with variables of cardiovascular function, markers of inflammation and telomere length. A national study cohort of 19 (median age 22, range 16-30 years) long-term (>10 years) HR NBL survivors was studied and the findings were compared with 20 age- and sex-matched controls. Frailty was defined as ⩾3 of the following conditions: low muscle mass, low energy expenditure, slow running and weakness. The prevalence of frailty was significantly higher among the HR NBL survivors 9/19 (47%) than among the controls (0%). Thirteen (68%) of the survivors reported significant physical health limitations in vigorous activities, as opposed to none of the controls. The HR NBL survivors had significantly shorter telomere length and higher serum levels of high sensitivity C-reactive protein than did the controls. Frail health and poor physical functioning are prevalent among HR NBL survivors and suggest premature aging. Survivors with gonadal damage, very low fat mass percentage, low glycosylated hemoglobin A1c and increased common carotid artery intima-media thickness may be more prone to early aging after high dose therapy.

State-of-the-art fertility preservation in children and adolescents undergoing haematopoietic stem cell transplantation: a report on the expert meeting of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) in Baden, Austria, 29-30 September 2015.

Nowadays, allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a well-established treatment procedure and often the only cure for many patients with malignant and non-malignant diseases. Decrease in short-term complications has substantially contributed to increased survival. Therefore long-term sequelae are reaching the focus of patient care. One of the most important risks of stem cell transplant survivors is infertility. As well as in the field of allo-HSCT also the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation in both boys and girls. Access to these procedures as well as their financing differs significantly throughout Europe. As all European children and adolescents should have the same possibility, the Paediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation organised an expert meeting in September 2015. This manuscript describes the recommendations for the diagnosis and pre-emptive procedures that should be offered to all children and adolescents in Europe who have to undergo an allo-HSCT.

Fertility preservation issues in pediatric hematopoietic stem cell transplantation: practical approaches from the consensus of the Pediatric Diseases Working Party of the EBMT and the International BFM Study Group.

Fertility preservation is an urgent challenge in the transplant setting. A panel of transplanters and fertility specialists within the Pediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation (EBMT) and the International BFM Study Group provides specific guidelines. Patients and families should be informed of possible gender- and age-specific cryopreservation strategies that should be tailored according to the underlying disease, clinical condition and previous exposure to chemotherapy. Semen collection should be routinely offered to all postpubertal boys at the diagnosis of any disease requiring therapy that could potentially impair fertility. Testicular tissue collection might be offered to postpubertal boys; nevertheless, its use has been unsuccessful to date. Oocyte collection after hormonal hyperstimulation should be offered to postpubertal girls facing gonadotoxic therapies that could be delayed for the 2 weeks required for the procedure. Ovarian tissue collection could be offered to pre-/post-pubertal girls. Pregnancies have been reported after postpubertal ovarian tissue reimplantation; however, to date, no pregnancy has been reported after the reimplantation of prepubertal ovarian tissue or in vitro maturation of pre-/post-pubertal ovarian tissue. Possible future advances in reproductive medicine could change this scenario. Health authorities should prioritize fertility preservation projects in pediatric transplantation to improve patient care and quality of life.

Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia.

A rat T-cell leukemia model was used to study the safety of germ cell transplantation as a mean of preventing infertility in males undergoing gonadotoxic cancer treatment. Donor germ cells were harvested from the testes of terminally ill leukemic rats and were either used directly or cryopreserved and thawed before transplantation by rete testis microinjection. All rats transplanted with testicular cells from leukemic donors developed signs of terminal rat T-cell leukemia, whereas control animals remained healthy. Cryopreservation of the donor germ cells caused a 3- to 6-day delay in the terminal phase of leukemia. When a known number of leukemic cells were mixed with germ cells and microinjected into the testis, the rate of appearance of terminal leukemia was directly related to the number of transferred leukemic lymphoblasts. As few as 20 leukemic cells were able to cause a cancer relapse resulting in terminal leukemia 21 days after transplantation in three of five transplanted animals. Our results demonstrate that germ cell transplantation with the presently used techniques is not safe enough for clinical use. Improved methods for purging testicular specimens of cancer cells or totally new approaches with transient xenogenetic host models to detect contamination of malignant cells must be developed before this technique can be offered to patients without fear of disease relapse.

Amifostine protects against early but not late toxic effects of doxorubicin in infant rats.

The improved prognosis and increased expected lifetime among long-term survivors of childhood malignancies have made these patients especially sensitiveto the late toxicity of cancer therapy and prone to secondary malignancies. Recently, new strategies aiming to protect against cancer treatment toxicity have been developed, including the drug amifostine (Ethyol), which is suggested to protect normal tissues from the toxic effects of radiation and cytotoxic agents. In the present study, the possible protective effect of amifostine against toxicity induced by a single injection of doxorubicin (3 mg/kg) in immature rats was evaluated. Specifically, we evaluated the protection against long-term toxicity and the effects of amifostine on growing immature tissues. Amifostine (50-200 mg/kg) given 15 min before doxorubicin had a significant protective effect against doxorubicin-induced early alopecia in young rats. Significant protection against cataract formation was obtained by the use of low-dose amifostine (50 mg/kg). However, amifostine did not protect young rats against the late toxic effect of doxoubicin on linear growth, body weight, plasma leptin levels, and heart or testicular tissue. Worrisome, and in contrast to earlier studies in adult rats, an increased doxorubicin toxicity actually was observed and mortality was increased when the higher doses of amifostine (100-200 mg/kg) were used. The present results suggest that more data from growing immature animal models are needed to analyze the safety of amifostine treatment and its mechanisms of action before wider clinical use of this drug in pediatric cancer patients is recommended.

Reactions of Leydig cells and blood vessels to lymphoblastic leukemia in the rat testis.

The testis is the third common site of relapse in childhood acute lymphoblastic leukemia (ALL). Despite the apparent clinical importance of testicular relapse, its pathogenesis is still unknown. The studies with an animal model of ALL have shown that many testicular factors are able to control the intratesticular infiltration and proliferation of leukemic lymphoblasts during the untreated course of ALL. In the present study, the ultrastructure of rat testicular interstitium infiltrated by leukemic lymphoblasts was studied in two groups of rats transplanted with rat T cell leukemia in early and late puberty. In both groups most of the leukemic cells infiltrating testicular interstitium were totally or partly enveloped by one or more Leydig cells, and the endothelial cells of capillaries, arterioles and venules were hypertrophic. The Leydig cells of the younger experimental group were by nuclear and cytoplasmic ultrastructure similar to the undifferentiated Leydig cells normally seen on the third postnatal week. The results suggest that Leydig cells bind leukemic lymphoblasts on their surface in vivo as also previously observed in vitro, and that ALL may disturb the pubertal maturation of Leydig cells. The occlusion of arterial and capillary lumina by folds of hypertrophic endothelial cells together with adhered leukemic lymphoblasts may impair the circulation of leukemic testes.

Regulation of testicular infiltration in acute lymphoblastic leukaemia of the rat.

The testis is a common site of relapse in childhood acute lymphoblastic leukaemia (ALL). In adults, testicular relapses of ALL are very rare. A similar age-difference in the frequency of the testicular infiltration exists also in the rat T-cell leukaemia. In the present investigation, the effect of various hormonal treatments and unilateral cryptorchidism on the form of testicular infiltrates by the rat T-cell leukaemia was studied. Inhibition of testicular activity by estradiol treatment (E2) of early pubertal rats injected i.p. with rat T-leukaemic lymphoblasts significantly decreased the proportion of the testis occupied by leukaemic infiltrates. The proportion of the testis occupied by leukaemic infiltrates was significantly higher in the abdominal testes of both early and late pubertal unilaterally cryptorchid rats, than in the scrotal testes of leukaemic control rats. Daily treatment of early pubertal rats with human chorionic gonadotrophin (hCG), or human menopausal gonadotrophin (hMG), did not have an effect on testicular leukaemic infiltration. These studies demonstrate that the leukaemic infiltration of the testis is influenced by the changes in the physiological activity of the testes.

A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry.

Accurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and γH2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.

Adverse health events and late mortality after pediatric allogeneic hematopoietic SCT-two decades of longitudinal follow-up.

Treatment-related late toxicities after pediatric allogeneic hematopoietic SCT (allo-HSCT) are increasingly important as long-term survival has become an expected outcome for many transplanted children and adolescents. In a retrospective cohort study, we assessed long-term health outcomes in 204 allo-HSCT survivors transplanted in childhood or adolescence (<20 years) between 1978 through 2000 after a median follow-up time of 12 (range 4-28) years. Data on conditioning regimen, adverse health events (AE) and growth and hormonal substitutions (hormone replacement therapies (HRTs)) were obtained from medical records. AEs were graded retrospectively according to Common Terminology Criteria for Adverse Events v3.0. Late deaths (⩾48 months after allo-HSCT) were evaluated separately. Multivariate analysis demonstrated that chronic GVHD (P<0.000) and longer follow-up time (P<0.05) correlated with AEs, whereas CY-based conditioning was inversely correlated (P<0.002). TBI and longer follow-up duration predicted more severe AEs (P<0.001 and P<0.001, respectively). HRTs were more frequent after TBI. Diabetes type II, dyslipidemia and hypertension were detected in 9, 7 and 7% of the survivors, respectively. Late deaths (n=22) were most frequently due to pulmonary failure (n=7), followed by secondary malignancy (n=5). The occurrence of AEs after pediatric allo-HSCT is high and likely to increase during extended follow-up, particularly in patients who have received TBI.

Autoantigenicity of the basal compartment of seminiferous tubules in the rat.

The expression of testicular autoantigens has been thought to be restricted to the luminal compartment of the seminiferous tubules. In the present study it was observed that germ cells in the basal compartment of seminiferous tubules and lamina propria of the seminiferous tubule bound antibodies in anti-testis immunosera in vivo after passive transfer of the sera by intra-peritoneal injection. The anti-testis immunosera were prepared in orchiectomised rats to avoid pre-collection adsorption of antibodies in the basal compartment of the seminiferous tubules. When testis-immune sera collected from non-orchiectomised rats or normal sera were transferred no such binding of antibodies to the basal germ cell surface or the lamina propria occurred. In Western blot analysis the anti-testis immune sera prepared in orchiectomised rats defined polypeptides from the adult rat testis with relative MWs of 19-23, 26-28, 30, 34, 38, 40-43, 45-47, 51-52, 56-57, 68, 78 and 97 kDa. The 40-43 kDa band was not detected by sera prepared in non-orchiectomised rats, suggesting that this autoantigen was expressed in the basal compartment of the seminiferous tubules. These observations suggest that the segregation of testicular autoantigens in the luminal compartment of seminiferous tubules is not complete for all of them in the rat and emphasize the role of more dynamic mechanisms in prevention of anti-germ cell autoimmune disease.

Ontogeny of immunosuppressive activity, MHC antigens and leukocytes in the rat testis.

The levels of immunosuppressive activity and the presence of MHC antigens and leukocytes were studied in the immature and the sexually mature rat testis. The immunosuppressive activities were measured from high-molecular weight (greater than 5 kDa) fractions of testis extracts using the protectin bioassay. The presence of MHC antigens and leucocytes was studied using the indirect immunoperoxidase method. In the immature rats, clusters of class I MHC antigen positive cells and a few cells expressing class II MHC antigen were present in the testicular interstitium. In the sexually mature rats, all the cells were MHC I+, and MHC II+ cells were numerous in the testicular interstitium. The seminiferous epithelium was MHC-negative in both the immature and the sexually mature testis. W3/25+ leukocytes were present in the interstitium and the tubular wall in both the immature and the sexually mature rat testis, but not in the seminiferous epithelium at any age. At 20-30 days of age, the testicular extracts were neutral or slightly stimulated 3H-TdR incorporation into peripheral blood lymphocytes, but at 44-60 days of age they inhibited lymphocyte proliferation significantly. In gel filtration, a peak of immunosuppressive activity was observed at approximately 400 kDa (protectin A) in both 20- and 60-days-old rat testes. A smaller peak was present at approximately 200 kDa in both age groups. This study shows that the testicular immunoregulatory microenvironment is different in the immature and the sexually mature rats. This may be important in such age-dependent human diseases as mumps orchitis and the testicular relapses of acute lymphoblastic leukemia.

Immunosuppressive activity in the rat seminiferous tubules.

Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.

Role of transforming growth factor beta in testicular immunosuppression.

The potential role of transforming growth factor beta (TGF beta) in the regulation of the immunological milieu of the testis was investigated. Antibodies neutralizing TGF beta reversed the previously observed suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract. Recombinant TGF beta 1 dose-dependently inhibited testicular interleukin-1-like factor-driven proliferation of murine thymocytes and ConA-stimulated rat peripheral blood mononuclear cells. Extracts of seminiferous tubules contained a M(r) approximately 25 K TGF beta-like growth inhibitor of the CLL-64 mink lung epithelial cell line. The present findings suggest an important role for TGF beta in testicular immunosuppression.

Effects of testicular cytokines on proliferation of rat T-leukaemic lymphoblasts in vitro.

A rat acute lymphoblastic leukaemia (ALL) model was used to study the mechanisms involved in the tendency to testicular relapse of ALL in boys. Previous studies have indicated that the infiltration and growth of leukaemic lymphoblasts in the testis are influenced by the same endocrine and paracrine control systems that regulate normal testicular function. In the present study the effects of aqueous extracts of scrotal, abdominal and estrogen-treated postpubertal rat testes on rat-leukaemic lymphoblast proliferation were evaluated. The effects of recombinant cytokines analogous to those observed in the testis on leukaemic cell DNA-synthesis were also evaluated since changes in the levels of these factors have been observed in association with cryptorchidism and low levels of gonadotropins. Transforming growth factor-beta 1 (TGF-beta1), significantly inhibited the proliferation of leukaemic rat lymphoblasts after 24 h of culture, whereas TGF-beta 2, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6 or combinations of them were inactive. Extracts of estrogen-treated testes and abdominal testes of unilaterally cryptorchid animals inhibited leukaemic T-cell proliferation significantly more than extracts of normal testes. The inhibitory activity in abdominal testes could be neutralized by anti-TGF-beta 1 antibodies. These results suggest that testicular TGF-beta 1 may influence growth of leukaemic lymphoblasts in the testis but also that other as yet unknown, testicular factors are involved in the regulation of leukaemic cell function in the testis.

Disturbances in craniofacial morphology in children treated for solid tumors.

Because of increased survival rates in childhood cancer, special interest has been focused on the side-effects of the therapy and the quality of life in long-term survivors. Our aim was to investigate craniofacial growth in children who had received different kinds of antineoplastic therapies for solid tumors. A total of 40 children treated in the Turku University Central Hospital were examined and divided into three different groups. Group 1 comprised 18 children treated for intracranial tumors with cranial irradiation (CRI) and chemotherapy (CT) including alkylating agents. Seven children out of 18 in this group received growth hormone (GH) therapy. In Group 2, 11 children with extracranial solid tumors also received multiagent CT including alkylating agents, but no CRI. Group 3 consisted of 11 children treated for Wilm's tumor with CT, which did not include alkylating agents or CRI. A total of 19 linear and four angular variables from the lateral cephalograms of the subjects were measured. Most deviations in craniofacial structures were found in children treated with combined CRI and multiagent CT. All disturbances were seen in the vertical measurements which were reduced when compared to the matched controls. It seems reasonable to assume that impaired growth following combined radio- and chemotherapy, as well as GH treatment, particularly affects cartilage-mediated growth. However, the deviations seen in the present study were fairly minor and did not usually require clinical consideration.

Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse.

The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on testicular morphology were explored. Micronucleus induction was observed at 10(-7) M concentration of DES and 17 beta-estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17 beta-estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes.