Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes.

Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children.

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes.

AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.

Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples.

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

Effect of Previous Alkylating Agent Exposure on Follicle Numbers in Cryopreserved Prepubertal and Young Adult Ovarian Tissue after Long-Term Xenografting.

PURPOSE AND METHODS: To elucidate whether previous cancer treatment affects graft recovery and follicle numbers, morphology, and development in grafts, cryopreserved ovarian biopsies obtained from 18 cancer patients aged 1-24 years with and without exposure to chemotherapy were xenografted as 1 mm3 fragments to immunodeficient mice for 22 weeks with exogenous stimulation. RESULTS: Graft recovery showed no association with chemotherapy exposure, pubertal stage, or leukemia contamination. Total follicle number per recovered graft varied between 0 and 1031 in the chemotherapy-exposed and between 0 and 502 in the non-chemotherapy-exposed group. Atretic follicles formed the largest proportion of the follicle pool in chemotherapy-exposed grafts. Increased atresia correlated with exposure to alkylating agents (mean ± SD 8866.2 ± 9316.3 mg/m2) but not with anthracyclines, pubertal stage, or leukemia contamination. CONCLUSION: The observation confirms the harmful effects of alkylating agents on ovarian tissue. Therapy at the median cumulative dose of 8866 mg/m2 leads to the decreased quality of cryopreserved ovarian follicles in children and young adults.

The effects of perfluorooctanoic acid (PFOA) on fetal and adult rat testis.

Perfluorooctanoic acid (PFOA) is a widely dispersed synthetic chemical, which accumulates in living organisms and has been connected with male reproductive disorders. To monitor the effects of PFOA, fetal rat testes or seminiferous tubule segments (stage VII-VIII) of adult rats were cultured in 0-100 µg/ml PFOA for 24 h. Afterwards, cAMP, progesterone, testosterone and StAR protein levels were measured from the fetal testes culture. Measurements were combined with immunohistochemistry, immunofluorescence, TUNEL and flow cytometric analysis to monitor cell death in somatic and germ cells. This study shows that the levels of cAMP, progesterone, testosterone and expression of StAR decreased significantly in PFOA 50 and 100 µg/ml. PFOA affected cell populations significantly by decreasing the amount of diploid, proliferating, meiotic I and G2/M-phase cells in adult rat testis. However, PFOA did not affect fetal, proliferating or adult rat Sertoli cells but an increased tendency of apoptosis in fetal Leydig cells was observed.

Retinoblastoma protein represses E2F3 to maintain Sertoli cell quiescence in mouse testis.

Maintenance of the differentiated state and cell cycle exit in adult Sertoli cells depends on tumor suppressor retinoblastoma protein (RB, also known as RB1). We have previously shown that RB interacts with transcription factor E2F3 in the mouse testis. Here, we investigated how E2f3 contributes to adult Sertoli cell proliferation in a mouse model of Sertoli cell-specific knockout of Rb by crossing these mice with an E2f3 knockout mouse line. In the presence of intact RB, E2f3 was redundant in Sertoli cells. However, in the absence of RB, E2f3 is a key driver for cell cycle re-entry and loss of function in adult Sertoli cells. Knockout of E2f3 in Sertoli cells rescued the breakdown of Sertoli cell function associated with Rb loss, prevented proliferation of adult Sertoli cells and restored fertility of the mice. In summary, our results show that RB-mediated repression of E2F3 is critical for the maintenance of cell cycle exit and terminal differentiation in adult mouse Sertoli cells.

Serum 25-Hydroxyvitamin D Concentrations at Birth in Children Screened for HLA-DQB1 Conferred Risk for Type 1 Diabetes.

CONTEXT: Vitamin D has several effects on the immune system that might be of relevance for the pathogenesis of type 1 diabetes (T1D). OBJECTIVE: To evaluate whether umbilical cord serum concentrations of 25-hydroxy-vitamin D (25[OH]D) differ in children developing either islet autoimmunity (IA) or overt T1D during childhood and adolescence. DESIGN: Umbilical cord serum samples from 764 children born from 1994 to 2004 with HLA-DQB1 conferred risk for T1D participating in the Type 1 Diabetes Prediction and Prevention Study were analyzed for 25(OH)D using an enzyme immunoassay. SETTING: DIPP clinics in Turku, Oulu, and Tampere University Hospitals, Finland. PARTICIPANTS: Two hundred fifty children who developed T1D diabetes at a median age of 6.7 years (interquartile range [IQR] 4.0 to 10.1 years) and 132 additional case children who developed IA, i.e., positivity for multiple islet autoantibodies. Cases were matched for date of birth, gender, and area of birth with 382 control children who remained autoantibody negative. The median duration of follow up was 9.8 years (IQR 5.7 to 13.1 years). MAIN OUTCOME MEASURE: The median 25(OH)D concentrations. RESULTS: The median 25(OH)D concentration in cord serum was low [31.1 nmol/L (IQR 24.0 to 41.8); 88% <50 nmol/L], but not statistically different between children who developed T1D or IA and their control groups (P = 0.70). The levels were associated mainly with geographical location, year and month of birth, age of the mother, and maternal intake of vitamin D during pregnancy. CONCLUSIONS: The 25(OH)D concentrations at birth are not associated with the development of T1D during childhood.

Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures.

Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Testes were cut into small pieces which were cultured atop agarose stands, using Minimum Essential Medium alpha supplemented with different supplements; melatonin, Glutamax, and different concentrations of KSR. The results showed that the duration of culture beyond 18 days had an impact on the number of differentiated germ cells. Supplementation with melatonin and Glutamax revealed a positive influence on the efficiency of male germ cell differentiation in vitro. Furthermore, the results confirmed that KSR had a positive effect on germ cell maturation and testosterone production, with a concentration of at least 10%. In conclusion, this study emphasizes the beneficial role of at least 10% KSR in the IVM of germ cells.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

Minimal residual disease of leukemia and the quality of cryopreserved human ovarian tissue in vitro.

Auto-transplant of cryopreserved ovarian tissue in leukemia patients carries a risk to reintroduce malignant cells. Maturation of ovarian follicles in vitro is a promising strategy to overcome the leukemic cell contamination. The follicle development and survival in 14 cryopreserved ovarian tissues with leukemia-specific PCR marker was evaluated after 7 or 14 days culture. Minimal residual disease (MRD) quantification was assessed by real-time quantitative PCR in order to identify the MRD positive (n = 6) and negative (n = 8) samples and to monitor levels of MRD before and after culture. The morphology of ovarian follicles were studied by light microscopy. After culture, no statistical significant differences were detected in follicle densities between MRD positive- and negative samples. Ovarian MRD either decreased below undetectable or fluctuated near the baseline level after 7 and 14 days in culture. This study provides quantitative in vitro evidence that leukemia contamination does not affect the follicle survival in cryopreserved ovarian tissue.

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro.

BACKGROUND: Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance. MATERIALS: In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested. RESULTS: Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles. CONCLUSION: This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.

Testicular function, semen quality, and fertility in young men after renal transplantation during childhood or adolescence.

BACKGROUND: This study was conducted to assess reproductive endocrine function, testicular volume, semen quality, and fertility in adult male patients after renal transplantation (RTx) during childhood or adolescence. METHODS: Twenty-four RTx recipients (median age: 28.1 years) were examined at a median of 18.6 years after RTx. Reproductive hormone levels and semen samples were studied and compared to those of 56 age-matched controls (median age: 30.2 years). Eight RTx men (33%) had been treated with cyclophosphamide. Medical records were retrospectively reviewed and data on puberty, dialysis, and immunosuppressive medication were analyzed to evaluate the prospect of fertility and sexual health. RESULTS: The testicular volumes and total sperm counts of the RTx patients were smaller than those of the controls with a median of 11.4 versus 33.9 mL, P<0.001 and 1.3 versus 135.5 million, P<0.001, respectively. Only four (22%) of the RTx men had normospermia in semen sample. The reproductive hormone levels were normal in the majority of the RTx survivors, with only two survivors showing androgen deficiency. Patients without history of cyclophosphamide therapy had significantly smaller testes and total sperm counts (median: 12.5 mL and 16.3 million) in comparison with their healthy peers (P<0.001 in both), and those with previous cyclophosphamide treatment showed further worse outcome (8.5 mL and 0 million, respectively, P<0.001 in both). CONCLUSIONS: Testicular function is often impaired even years after transplantation and poor semen quality decreases the prospect of fertility in men after pediatric RTx.

Autologous ectopic grafting of cryopreserved testicular tissue preserves the fertility of prepubescent monkeys that receive sterilizing cytotoxic therapy.

Boys faced with future sterility as a result of the need of a sterilizing cancer therapy might avoid this fate by engraftment of cryopreserved immature testicular tissue after therapy is completed. Efforts to address this important survivorship issue have been encouraged by reports of the long-term survival and proliferation of human spermatogonia after xenotransplant of cryopreserved immature testicular tissue into immunocompromised murine hosts. However, spermatogenic arrest at the pachytene spermatocyte stage that occurs in this situation has been associated with a failure in sperm production. In this study, we used a prepubescent simian model to address the possibility that testicular tissue engraftment is insufficiently supported in the model to allow suitable maturation of germ cells. Briefly, we carried out autologous orthotopic grafting of cryopreserved testicular tissue from four prepubescent monkeys and one pubescent rhesus monkey after testicular irradiation and castration of the host animal. Five months after implantation of scrotal grafts, we determined that 3% to 7% of the autografts could be recovered with spermatogenesis proceeding through spermatozoa formation in 13% to 17% of the seminiferous tubules formed in the grafts. In contrast, Sertoli cell-only tubules were detected in parallel xenografts transplanted into immunocompromised mice. Our results show that cryopreservation of testicular tissue from prepubescent primates can maintain the fully functional capacity of spermatogonia to produce sperm, but that host conditions are critical for spermatogenic maturation. Furthermore, our results establish an initial perspective on the quantity of cryopreserved material needed to ensure success in preserving fertility through testicular tissue grafts.

Peritubular myoid cells have a role in postnatal testicular growth.

FSH stimulates testicular growth by increasing Sertoli cell proliferation and elongation of seminiferous cords. Little is known about the peritubular myoid cells in testicular development. In order to investigate the role of peritubular myoid cells in early testicular growth in rodents, two traditional models to induce testicular growth were used: FSH treatment and hemicastration. In order to affect proliferation of peritubular myoid cells, both treatments were combined with imatinib, a tyrosine kinase inhibitor. In addition, effects of imatinib on human testicular peritubular cell proliferation were investigated. Testicular weight, diameter and length of seminiferous cords, numbers of germ, Sertoli and BrdU-positive cells and FSH-levels were measured. FSH treatment and hemicastration increased length of the seminiferous cords and testicular weight by increasing first the early proliferation of peritubular myoid cells and later also the proliferation of the Sertoli cells. Imatinib blocked the FSH and hemicastration -induced testicular hypertrophy and decreased the proliferation of PDGF-stimulated human testicular peritubular cells in vitro. Present results provide new evidence that peritubular myoid cells have an important role in postnatal testicular growth.

Hedgehog signalling promotes germ cell survival in the rat testis.

Hedgehog (Hh) signalling has a crucial role in testis development. Sertoli cell-derived desert hedgehog (DHH) guides the formation of testis cords and differentiation of foetal-type Leydig cells. Dhh mutant mice are infertile due to a block in germ cell differentiation, hypogonadism and hypoandrogenism. Hh signalling pathway components are also expressed in postnatal testis. In the rat testis the transcription factor of the Hh pathway, glioma-associated oncogene homologue (GLI1), is expressed by a wide variety of germ cells. This suggests that Hh signalling is involved in spermatogenesis at many different levels. Our data show that canonical Hh signalling is turned off in early condensing spermatids that strongly express the negative regulator of the pathway, suppressor of fused (SUFU). Most of the Hh pathway specific mRNAs display the highest values in stages II-VI of the rat seminiferous epithelial cycle. The key endocrine regulator of germ cell differentiation, FSH, down-regulates Dhh mRNA levels in vitro. Hh signalling inhibition in vitro leads to massive apoptosis of germ cells. In prepubertal rat testis imatinib mesylate-induced inhibition of tyrosine kinases impinges on Dhh transcript levels and Hh signalling. Our data indicate that Hh signalling is part of the paracrine signalling network in the rat testis. It promotes the survival of germ cells and is suppressed by FSH.

Testicular failure and male infertility in the monogenic Mulibrey nanism disorder.

CONTEXT: Few monogenic mutations causing human male infertility have been identified to date. OBJECTIVE: We studied pubertal development and fecundity in males with Mulibrey nanism (MUL) caused by mutations in the TRIM37 gene. DESIGN, SETTING, AND PATIENTS: Twenty-eight male MUL patients of the Finnish national cohort aged 8.7 to 50.0 yr (median age, 28.8) at the end of observation were followed for 10 yr beginning from 2000-2001. MAIN OUTCOME MEASURES: Clinical characteristics, reproductive hormone levels, semen quality, and testicular histology were assessed. RESULTS: The external genital phenotype was normal. In childhood and prepuberty, serum levels of FSH, LH, testosterone (T), and inhibin B were normal. Puberty started spontaneously at a median age of 12.6 yr (range, 11.1-15.0), and FSH, LH, T, and inhibin B levels increased adequately until midpuberty. Thereafter, testicular growth and virilization proceeded slowly. Concomitantly, FSH, and to a lesser extent LH, showed a progressive increase to hypergonadotropic levels in all patients, whereas inhibin B decreased and T leveled off. Testicular size was small (median volume, 8.7 ml; range, 3.5-18.3 ml in adults). All semen samples showed severe oligoasthenozoospermia or azoospermia. None of the patients had a history of spontaneous fertility, but four men had undergone infertility treatment, which in one case was successful. All histological MUL samples showed varying degrees of degeneration. CONCLUSIONS: All adult MUL males have a unique disorder of testicular function with small testes, elevated FSH and LH, and low inhibin B. In MUL, mutations in TRIM37 lead to disturbance of sexual maturation, and fertility is severely compromised. Thus, TRIM37 is a novel gene causing male infertility.

Testicular recovery after irradiation differs in prepubertal and pubertal non-human primates, and can be enhanced by autologous germ cell transplantation.

BACKGROUND: Although infertility is a serious concern in survivors of pediatric cancers, little is known about the influence of the degree of sexual maturation at the time of irradiation on spermatogenic recovery after treatment. Thus, we address this question in a non-human primate model, the rhesus monkey (Macaca mulatta). METHODS: Two pubertal (testis size 3 and 6.5 ml, no sperm in ejaculate) and four prepubertal (testis size 1 ml, no sperm in ejaculate) macaques were submitted to a single fraction of testicular irradiation (10 Gy). Unilateral autologous transfer of cryopreserved testis cells was performed 2 months after irradiation. Testicular volume, histology and semen parameters were analyzed to assess irradiation effects and testicular recovery. RESULTS: Irradiation provoked acute testis involution only in the two pubertal monkeys. Subsequently, testis sizes recovered and sperm was present in the ejaculates. Longitudinal outgrowth of seminiferous tubules continued, and, in testes without autologous cell transfer, 4-22% of tubular cross sections showed spermatogenesis 2 years after irradiation. In contrast, the four prepubertal monkeys showed neither a detectable involution as direct response to irradiation, nor a detectable growth of seminiferous tubules later. However, two of these animals showed spermarche 2 years after irradiation, and 8-12% of tubules presented spermatogenesis. One prepubertally irradiated monkey presented fast growth of one testis after cell transfer, and showed spermarche 1 year after irradiation. The infused testis had spermatogenesis in 70% of the tubules. The contralateral testis remained smaller. CONCLUSION: We conclude that irradiation before puberty has a severe detrimental effect on outgrowth of seminiferous tubules. But, within the seminiferous epithelium, spermatogenetic recovery occurs at a low rate with no detectable relation to the maturity of the epithelium at irradiation. We also show that autologous testis cell transplantation can enhance spermatogenesis, but only in isolated cases.